• Bacterial pathogenesis;
  • Virulence;
  • Signature-tagged mutagenesis;
  • Transposons


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Signature-tagged mutagenesis
  5. 3Identification by STM of the role of mycobacterial lipids in virulence
  6. 4Host tropism
  7. 5Mechanisms of survival and persistence within the host
  8. 6Crossing of the blood–brain barrier
  9. 7Concluding remarks
  10. Acknowledgements
  11. References

Studies on the genetic basis of bacterial pathogenicity have been undertaken for almost 30 years, but the development of new genetic tools in the past 10 years has considerably increased the number of identified virulence factors. Signature-tagged mutagenesis (STM) is one of the most powerful general genetic approaches, initially developed by David Holden and colleagues in 1995, which has now led to the identification of hundreds of new genes requested for virulence in a broad range of bacterial pathogens. We have chosen to present in this review, the most recent and/or most significant contributions to the understanding of the molecular mechanisms of bacterial pathogenicity among over 40 STM screens published to date. We will first briefly review the principle of the method and its major technical limitations. Then, selected studies will be discussed where genes implicated in various aspects of the infectious process have been identified (including tropism for specific host and/or particular tissues, interactions with host cells, mechanisms of survival and persistence within the host, and the crossing of the blood brain barrier). The examples chosen will cover intracellular as well as extracellular Gram-negative and Gram-positive pathogens.


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Signature-tagged mutagenesis
  5. 3Identification by STM of the role of mycobacterial lipids in virulence
  6. 4Host tropism
  7. 5Mechanisms of survival and persistence within the host
  8. 6Crossing of the blood–brain barrier
  9. 7Concluding remarks
  10. Acknowledgements
  11. References

Pathogenic bacteria have evolved complex molecular mechanisms to invade and survive within their hosts. One can define a virulence gene as a gene whose product is necessary for survival and persistence within the host [1]. Virulence factors in bacterial pathogens can act either directly on the infectious process, like toxins and adhesins, or indirectly, by participating in regulatory processes or because they are required for bacterial survival. Since virulence genes participate in various stages of the infectious process, their inactivation may lead in some cases to a complete loss of virulence or more generally to intermediate phenotypes corresponding to variable degrees of attenuation. A number of genetic methods like signature-tagged mutagenesis (STM) or in vivo expression technology (IVET) were developed to discover such new genes, especially those that cannot be identified by computer-assisted genomic predictions, or by subtractive DNA–DNA hybridization techniques (see for example [2]). STM, initially described by the group of David Holden [3], has now been applied to a variety of bacterial pathogens. The studies published over the past 10 years establish that STM is one of the most powerful and versatile large-scale genetic approaches to identify virulence determinants and can be therefore considered as a functional genomic approach.

However, careful examination of the publications reveals a very important qualitative and quantitative heterogeneity of the data. This heterogeneity is in part due to the variety of the organisms studied, of the models, and sizes of the screens. Therefore, we revaluated the data from STM studies, focusing on those that provided the most significant information on the role of the genes involved in bacterial pathogenicity.

We will first recall below the major parameters that need to be set-up to perform an efficient STM screen and some of the restraints that may hinder the identification of attenuated candidates. The general features of the mutants identified through STM carried out on Gram-positive and Gram-negative microorganisms will be summarized. Then, selected examples will be organized into four categories: (i) STM studies on Mycobacterium tuberculosis which provided, unlike most other STM studies, overlapping information on the role of lipid biosynthesis in mycobacterial virulence; (ii) STM screens performed in several Gram-positive and Gram-negative pathogens (Streptococcus pneumoniae, Staphylococcus aureus and members of the genus Yersinia) where pathogenicity was evaluated simultaneously in different hosts or tissues, to understand the molecular bases of host tropism; (iii) studies addressing the mechanisms of survival and persistence within the host, including adaptation to stress and nutritional deficiencies, and factors involved in colonization processes; and finally, (iv) STM screens aimed at identifying genes responsible for the traversing of physiological barriers.

2Signature-tagged mutagenesis

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Signature-tagged mutagenesis
  5. 3Identification by STM of the role of mycobacterial lipids in virulence
  6. 4Host tropism
  7. 5Mechanisms of survival and persistence within the host
  8. 6Crossing of the blood–brain barrier
  9. 7Concluding remarks
  10. Acknowledgements
  11. References


Transposable elements have been widely used to study microorganisms [4]. STM is an evolution of traditional transposon mutagenesis that allows the large-scale analysis of transposon-insertion mutants for the identification of virulence genes in pathogenic bacteria. This method has two major advantages over other classical – targeted or random – gene inactivation approaches: (i) conceptually, STM is based on a negative selection of the mutants, i.e., mutants, which have lost the capacity to survive in a given host (see below), allowing the discovery of virulence genes without prior indication of their nature or function; (ii) technically, many mutants can be screened at the same time (the mutants assembled within pools are easily identified by a unique sequence – or tag – carried by the inserted transposon), which allows, in principle, a rapid and exhaustive analysis of virulence factors in a given organism. It is worth mentioning that the principle of applying STM does not necessarily require a transposon. DNA tags can be included during allelic replacement (signature-tagged allele replacement) on a systematic genome-wide scale [5]. This has been applied to Saccharomyces cerevisiae, also called bar-coding [6]. We will focus below on STM studies that used transposons.

STM is most often used for in vivo screening, although STM screens have also been performed on in vitro systems (like for example, in eucaryotic cell cultures, see Table 1). Briefly, in in vivo studies, pools of inoculation are constituted and administered (by a given route) to the animal model chosen (see Fig. 1). Bacteria are recovered from the target organs at selected intervals after infection. By dot-blot hybridization techniques with PCR-amplified tags, it is possible to identify the non-attenuated mutants present in the pool recovered and, thus, to infer which mutants were unable to persist in vivo. Determination of the transposon insertion site provides then the direct identification of the gene that has been inactivated.

Table 1.  Summary of published STM studies
 Animal model (or cell type): target organInoculation (route)Pool sizeNo. of attenuated mutants1/No. candidates2/No. of mutants tested3Reference
  1. I.M.: intramuscular, I.N.: intranasal; I.P.: intraperitoneal; I.V.: intravenous; I.T.: intratracheal; S.C.: subcutaneous; P.P.: Peyer's patches; T.U.: transurethral; NP: not published; NS: not specified. No. of attenuated mutants1: Mutants shown to be attenuated after individual test in the given model. No. of candidates2: Potentially attenuated candidates, selected by dot blot analysis (i.e., clones that were not recovered from the output pools or PREC). No. of mutants tested3: total number mutants tested. Several examples are detailed below. They reflect the heterogeneity, and in some cases, the lack of completion, of the different STM studies. A. pleuropneumoniae[8]: 71/1052/20643 means that out of 20643 mutants tested, 1053 candidates were identified. DNA sequence analysis of these candidates identified only 55 different genes. Of these 55 candidates, fourteen were individually tested for attenuation in vivo. Only 71 of them were actually attenuated. B. abortus[58]: 141/272/1783 means that out of 1783 mutants tested, 273 candidates were identified. These 27 candidates were individually tested for attenuation in vivo. Only 141 of them were actually attenuated. H. pylori[29]: >141/2522/9603 means that out of 9603 mutants tested, 2522 non-colonizing H. pylori mutants were isolated. DNA sequencing identified 47 different genes. Only 141 mutants were individually tested for attenuation in vivo. All of them were identified as heavily attenuated in the animal assay. N. meningitidis[68]: >221/2342/28503 means that out of 28503 mutants tested, 2342 candidates were identified. Only 221 mutants were tested in the animal model; all of them showed a defect. S. enterica (Dublin) [14]: >21/1–2%2/52803 means that out of the 52803 mutants tested, the number of attenuated candates was evaluated to 1–2%2 of the clones. Only 21 Spi-2 mutants were studied in details (insertion in the sseD and ssaT genes). V. cholerae[27]: 231/512/11003 means that out of 11003 mutants tested, 512 candidates were identified. Only 231 mutants showed a defect in the animal model. Y. pseudotuberculosis[25]: >141/312/6033 means that out of 6033 mutants tested, 312 candidates were identified. Twenty of these candidates were tested in vivo. Only 141 of them sowed a significant attenuation of virulence. S. agalactiae[73]: >161/1202/16003 means that out of 16003 mutants tested, 1202 candidates were identified. Ninety-two of them were sequenced, among which only 161 were individually tested in the animal model. All the mutants showed a (modest to high) reduction of virulence. S. pneumoniae[23]: 251/1862/17863 means that out of 17863 mutants tested, 1862 candidates were identified. Fifty-six mutants were sequenced, leading to the identification of 47 different genes. Only 251 of them were individually tested in vivo, all resulting in a broad range of virulence defects. S. pneumoniae[21]: >161/3872/61493 means that out of 61493 mutants tested, 3872 candidates were identified. The site of Tn insertion was determined for 337 of them. Seventeen attenuated candidates were tested in the animal model. Only 161 of them showed a reduced virulence.

Gram-negative bacteria
A. pleuropneumoniaePig: lungI.T.4020/110/800[57]
A. pleuropneumoniaePig: lungI.T.487/105/2064[58]
B. aviumTurkey: tracheaI.T.24 NS* [59]
B. abortusMouse: spleenI.P.461427/178[60]
B. melitensisMouse: spleenI.P.9618/19/672[31]
B. melitensisMouse: spleenI.P.9636/37/1152[32]
B. suisMacrophages9614/32/1152[30]
B. pseudomalleiMouse: spleenI.P.961/1/96[61]
C. rodentiumMouse: colonOral24–2814/33/576[62]
E. coli K1HBMEC967/26/3360[53]
E. coli K1Rat: colonOral9616/18/2140[37]
E. coli K1Rat: colon, spleenOral/I.P.483/3/192[63]
E. coli uropathogenicMouse: kidney, bowelIntra-organ92/461/9/2049[38]
H. pyloriGerbil: stomachOral4014/252/960d[29]
K. pneumoniaeMouse: colon/Oral4813/44/2200[64]
 Human intestinal cells4816/59/2200 
K. pneumoniaeMouse: bowel/fecesIntra-organ/oral4816/19/1440[65]
L. pneumophilaGuinea pig: lung, spleenI.T.9616/16/1386[66]
L. pneumophilaAmoebes966/12/700[67]
N. meningitidisRat: bloodI.P.9622/234/2850[68]
N. meningitidisSerum44–4820/1594548[54]
 HUVEC44–4817/ 4548Pelicic, NP*
P. multocidaMouse: spleenI.P.902562/1710[69]
P. multocidaMouse: bloodI.P.425/15420[70]
 /Chicken/I.M /15/35420//
P. mirabilisMouse: kidney, bowelT.U.962/2/280[71]
P. aeruginosaRat: lungI.T.9613/331056[72]
S. enterica (Dublin)Mouse: spleenI.V.9621–2%/5280[14]
 Calf: spleen      
S. typhimuriumMouse: spleenI.P.964/40/1152[3]
S. typhimuriumMouse and Calf: P.P., spleenOral24–3016/16/260[13]
V. choleraeMouse: intestineOral4823/51/1100[27]
V. choleraeMouse: intestineOral96 164/9600[26]
Y. enterocoliticaMouse: spleenI.P.9655/682000[7]
Y. pseudotuberculosisMouse: spleenI.V.9614/31/603[25]
Y. pseudotuberculosisMouse: cecumOral4814/19/960[73]
Y. pestisMouse: spleenS.C.203/16/300[74]
Gram-positive bacteria and Mycobacteria
L. monocytogenesMouse: brainI.V.4818/602000[47]
 Mouse: liverI.V.48740/4176[49]
S. aureusMouse: wounds, abscesses, spleen, liverS.C.956237/1520[24]
 Mouse: spleen, bloodI.P.9616/50/1248[56]
S. agalactiaeRat: spleenI.P.8016120/1600[75]
S. pneumoniaeMouse: lungI.N.5088126/1250[22]
S. pneumoniaeMouse: spleen, lung,I.P./I.N9425/186/1786[23]
S. pneumoniaeMouse: lung, nasopharynx, bloodI.N./I.P6316/387/6149[23]
M. tuberculosisMouse: lungI.V.4816/79/1927[10]
M. tuberculosisMouse: lungI.V.483/14/576[11]

Figure 1. Generation and screening of the banks of mutants. (a) Constitution of the banks of mutants and screening strategy. The tagged transposon (48 in our example) are introduced individually into the desired bacterium to generate 48 banks of mutants; a, b and n represent the different clones within each bank (i.e., carrying the same tag). Pools of mutants, designated a, b, to n, are constituted taking one mutant from each bank. Mutants within a pool are grown individually. Tn mutants are assembled in order into microtiter wells according to their tag number [56], and then mixed. Quantities of each mutant are adjusted, to counterbalance growth variations. The pools are used to infect animals (pools of inoculation, PINO). After an appropriate period of infection, target organs are recovered from infected animals and homogenized (recovered pools, PREC). Chromosomal DNAs are prepared from PINOs and serve for PCR amplification with primers specific for the tags (corresponding to the conserved flanking regions). The random portions of each tag within the pool are labelled and used to hybridize membranes carrying all the tags and serves as a positive control (all the tags are hybridized). The same procedure is used for the PRECs. Bacteria that were eliminated during passage in the animal correspond thus to: a positive signal on the PINO membrane and a negative signal on the corresponding PREC membrane. (b) Virulence attenuated pools. Candidates selected after the first round may be re-used in a second assay to eliminate mutants giving false negative signals. The same protocol is used, increasing stringency and sensitivity of the assay.

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For each pathogen to be studied, several critical parameters must be carefully established to allow an efficient STM screen in vivo: (i) the transposon chosen for chromosomal mutagenesis should preferably insert at random in the chromosome, the presence of hot spots of insertion increasing considerably the number of mutants to be screened (very few transposons have no target site specificity and hence randomness is almost never achieved); (ii) the infecting pool size must be determined with respect to the inoculum dose (generally 48-96 mutants per pool), to ensure a good representation of each mutant in each pool; (iii) the route(s) of administration as well as the infecting dose(s) must also be carefully defined; and finally, (iv) the kinetics of bacterial survival (and/or multiplication) in the target organ(s) must be established for the wild-type strain, to determine when and where to evaluate a possible attenuation of virulence.

2.2Technical restraints

Signature-tagged mutagenesis is limited to finding non-essential genes (i.e., not required for growth in broth) in microorganisms that are transformable and/or allow the random insertion of transposon. Once an efficient and random transposition system has been set-up, and the major parameters of the in vivo model defined, other elements may alter the efficiency and/or reproducibility of the screening procedure. For example, despite all precautions in initial tag selection, some tags are not amplified from chromosomal DNAs of bacteria after their passage in the animal. This unexplained – but frequently encountered – problem of signal quality results in loss of reproducibility and to the identification of “false-negative” candidates. This technical limitation can be circumvented, or at least reduced, by using a procedure described by [7]. The assay consists of re-testing in a second screen, the candidates selected after a first screen by re-assembling them within new pools that in turn are injected to the animal. This procedure lengthens the duration of the experimentation, but may reduce appreciably the number of false-negative candidates.

In many published screens, some candidates are repeatedly found, suggesting a bias in the screening procedure. This bias may be due, at least in part, to the infection model. Indeed, the choice of a route of infection and/or of a target organ, will have an impact on the nature of the candidates, and probably highlight particular defects. The redundancy may also be due to the fact that transposons do not insert absolutely at random. In practical terms, this prevents an exhaustive genome-scale study, and can explain why very few complete STM study has been achieved. In addition, for human bacterial pathogens, evaluation of virulence requires an animal model that allows an accurate reproduction of the natural route of infection. Such a model is not always available or reliable, or the infecting dose is too low to allow constitution of large pools of mutants, thereby reducing the interest of the method. Finally, in vivo STM screens do not allow discriminating between the genes that are dispensable for growth in cellular models from those that are essential.

In spite of all these experimental difficulties and/or limitations, STM has been applied successfully to many bacterial pathogens. STM has been also applied to pathogenic fungi, and extensively to S. cerevisiae and toxoplasma [8,9]. From 1995 to 2002, more than 20 STM studies on pathogenic bacteria were completed and to date, more than 42 STM studies have been published. They relate to 22 different Gram-negative bacteria and five Gram-positive bacteria, including one in Mycobacterium (see Table 1).

2.3General features of the mutants identified by STM

We choose to discuss selected contributions of STM to the understanding of bacterial pathogenesis rather than to provide a systematic compilation of all the published studies. Nevertheless, we include an updated list of all the studies identified as STM screens of pathogenic bacteria published to date (Table 1) summarizing, for each pathogen: (i) the number of mutants tested, the number of attenuated candidates selected, and the number of attenuated mutants identified; (ii) the animal model used (or in vitro system) and the route of infection. As expected, a majority of studies identified previously known virulence genes, validating the concept of STM. However, it is worth noting that one finds few common genes between the different studies, even when performed on the same pathogen, reflecting the lack of exhaustivity of most published studies. This lack of overlap between studies is also artificially amplified by the fact that in some cases the criteria to define a virulence gene are only very preliminary. In several studies, impressive lists of potential virulence genes are provided, only few of which might be “genuine” virulence genes, i.e., genes encoding factors likely to be directly related to the infectious process like adhesins, invasins, cytolysins, toxins, effectors.

Generally, mutations in genes encoding surface molecules range from 25% up to 50% of the candidates identified by STM, highlighting their crucial role in pathogenesis. This is not surprising, since extracellular as well as intracellular bacteria must come into contact with host cells at some point of the infection. Surface molecules can participate in many different essential processes like: crossing of barriers, tropism, survival and/or multiplication, stress adaptation, colonization and dissemination. In the majority of STM studies, genes encoding elements of the cell wall were identified, including adhesins, transporters, as well as genes involved in peptidoglycan biosynthesis or modification. Membrane transporters were also shown to play a key role, acting as surface molecules dedicated to metabolite transport. STM screens also identified genes belonging to biosynthetic pathways, unravelling their importance during an infectious process when bacteria encounter environments that cannot provide essential nutrients. Regulatory genes were also identified. The controlled expression of “the right genes at the right time” is indeed essential to establish, maintain and/or develop the infection. The last category of genes identified by STM does not share any significant similarity with genes of known or suspected function; they can reach up to 50% of the candidates identified by STM.

3Identification by STM of the role of mycobacterial lipids in virulence

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Signature-tagged mutagenesis
  5. 3Identification by STM of the role of mycobacterial lipids in virulence
  6. 4Host tropism
  7. 5Mechanisms of survival and persistence within the host
  8. 6Crossing of the blood–brain barrier
  9. 7Concluding remarks
  10. Acknowledgements
  11. References

The wide array of complex lipids and lipoglycans displayed on its surface makes members of the genus Mycobacterium unique among bacterial pathogens. The mycobacterial cell envelope has long been thought to participate in pathogenicity but rigorous genetic proof was lacking. Bacterial multiplication, and concomitant tissue damage within the infected host, occurs primarily in the lung. Application of STM to M. tuberculosis was performed the same year by two different teams, using the lung as the target organ in the mouse model. Unlike most of other STM studies, and despite a relatively low number of mutants screened in each study (1927 [10] and 576 [11], respectively), in both screens, the attenuated mutants contained insertions in the same portion of the chromosome (Fig. 2). The region identified contains genes implicated in biosynthesis, transfer, export and insertion of various lipids, including phtiocerol dimycocerosate (PDIM) that is found only in pathogenic mycobacteria [11]. In both studies, transposon insertions were identified immediately upstream of fad26, in the promoter region of a large operon that includes ppsA–E genes, and in the mmpL7 gene (Fig. 2). In the first study [10], out of the sixteen transposon insertions producing an attenuated phenotype, four different insertions mapped in the 50 kb cluster. Insertions occurred just upstream from fad26 and within fad26, drrC and mmpL7 (the model for synthesis and export of PDIM presented in the lower part of Fig. 2 is adapted from [11]). Further biochemical analyses of strains carrying mutations in this locus revealed that mutants unable to synthesize or translocate PDIMs exhibited increased cell wall permeability. Thus, the cell envelope might also participate passively in microbial resistance to host defences and hence contribute to virulence. In the second study [11], 14 attenuated candidates were isolated. Most of the transposon insertions disrupted orf encoding proteins of unknown functions and three insertions occurred within a 44 kb region containing genes whose products are involved in the synthesis of PMID. The first insertion occurred immediately upstream of fad26; the second fell in fad28 and the third one, in mmpL7 MmpL7 is a member of a family of 13 predicted lipid transporters encoded by the genome of M. tuberculosis. These three mutants were further tested individually for alteration of virulence. The three mutant strains showed a severe growth defect in the lungs of infected mice (after iv infection). In contrast, multiplication was identical to that of the wild-type strain in the spleens and livers, indicating that PMID is specifically required for growth of M. tuberculosis in the lungs. More recently, Cox and co-workers [12] identified a new mmpL gene, mmpL8, by an extension of their previous STM screen. MmpL8 is essential for the biogenesis and export of a sulfated glycolipid, termed SL-1, to the mycobacertial surface. In addition to its role in SL-1 biosynthesis, MmpL8 is required to establish a high level of infection in mice. Since SL-1 itself is not required for acute infection in mice, it is likely that MmpL8 transports other molecules that are important for pathogenesis.


Figure 2. The mycobacterial envelope and biosynthetic genes. (Upper) Schematic organization of the PDIM locus. The arrows indicate the orientation and approximate size of the different genes. Transposon insertions are indicated as triangles: black triangles (upper) correspond to the insertions described in [11]; grey triangles (lower) to the insertions described in [10]. (Lower) Model for synthesis and export of the PDIM. Fad26 and Fad28 release phthiocerol and mycocerosic acids from their respective syntases (PpsA–E and Mas) and condense them to form PMID. PDIM, phthiocerol dimycocerosate; mAG, mycolyl-arabinogalactan layer; PG, peptidoglycan layer; CM, cytoplasmic membrane.

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Altogether, these results indicate that the peculiar nature of the mycobacterial cell wall plays an important role in pathogenesis.

4Host tropism

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Signature-tagged mutagenesis
  5. 3Identification by STM of the role of mycobacterial lipids in virulence
  6. 4Host tropism
  7. 5Mechanisms of survival and persistence within the host
  8. 6Crossing of the blood–brain barrier
  9. 7Concluding remarks
  10. Acknowledgements
  11. References

STM is an approach of choice to study a series of mutants in different models simultaneously, giving clues to explain host susceptibility to particular bacteria and helping to identify the factors implicated in the tropism for a given host.

4.1Salmonella pathogenicity islands

Several screens on Salmonella species suggested the existence of pathogenicity islands (PAIs) [13–15] and illustrated the interest of using the STM to lead comparative analyses in different animals hosts. The two largest pathogenicity islands (SPIs) in the serovar Typhimurium genome, designated SPI-1 and SPI-2, each encode a type III secretion system (TTSS) with structural homology to each other and to the TTSS of other known pathogens (reviewed in [16,17]). The SPI-1 TTSS is required for cell invasion, and it induces significant intestinal secretory and inflammatory responses [18]. The SPI-2 TTSS is required for intracellular replication and systemic infection. TTSSs comprise a specialized protein export apparatus that spans the inner and outer bacterial membranes and acts as the secretion machinery for bacterial effectors. A subset of the secreted effectors form a complex called the translocon. Upon infection, the TTSS translocates bacterial effector proteins across three membranes and delivers them directly into the target host cell, where they alter or initiate cytoskeletal rearrangements, signal transduction, and vesicular trafficking (reviewed in [19]). The aim of the work carried out by Tsolis et al. [13] was to look for genes involved in adaptation to different hosts, by screening 260 mutants of S. enterica serotype typhimurium in two different hosts, the calf and the mouse, after oral inoculation. Bacteria were recovered from the spleens and Peyer's patches of infected animals. Out of the 17 attenuated candidates identified, thirteen mutants were defective for colonization in both animal species; three were specific for mice and one for calves. Seven of the mutants defective for colonization of Peyer's patches and/or spleens of both host species carried insertions in previously known virulence determinants (including in the SPI-1 PAI; the rfa locus and the svp operon), validating the screening procedure. Four insertions were located in different genes located within either SPI-1 or SPI-2 PAI. One of the mutants showed an attenuation of virulence in the mouse, but not in the calf, suggesting a defect of colonization of the intestinal mucous membrane [13]. The inactivated gene, slrP (for Salmonella Leucine-rich Repeat Protein) is located in a third small PAI of 2.9 kb, and is similar to the ipaH and yopM genes, of Shigella flexneri and Yersinia pestis, respectively. The two corresponding proteins are secreted by TTSS, and share common features with the SlrP protein, including the presence of leucine-rich repeats. These data reinforce the conclusions about the importance of TTSS in colonization of the gastro-intestinal tract.

In the second comparative study on Salmonella enterica serovar Dublin, the authors tested a pool of mutants in calves (the natural host) and in mice, for the colonization of spleen [14]. They identified a strain carrying a mutation in the sseD gene, which encodes a predicted secreted protein. This gene belongs to the Spi-2 PAI, previously identified by Holden and co-workers [15] as necessary for the intravacuolar replication of Salmonella (see [20], for a review). The sseD mutant is defective for survival in calves, and does not allow induction of systemic or enteric infection. A second mutant, carrying a mutation in ssaT, is also located in Spi-2, and shows the same characteristics. These results demonstrated, for the first time, the participation of Spi-2 PAI in intravacuolar survival. These different studies suggest that each pathogenicity island would be implicated in a particular stage of bacterial pathogenesis, either in a sequential way and/or in a specific way, in response to particular host signals. Spi-2 has now become a major field of study by Salmonella researchers. The SPI-2-encoded TTSS is expressed during the intracellular stage of infection. A key SPI-2-dependent phenotype is the maturation of the vacuoles by selective interactions with the endosomal–lysosomal pathway and the formation of tubular membranous extensions called Salmonella-induced filaments [19].

4.2Multiple-model screening to study tissue tropism

In addition to host-specific tropism, tissue-specific tropism for a given host was also analyzed by STM. A study on S. pneumoniae screened hundreds of mutants in several models of infection [21]. S. pneumoniae is responsible for various infections, including pneumonia, otitis and meningitis. Of 6149 mutants analyzed in the lungs of infected mice, 387 were selected after two successive rounds of screening. Surprisingly, among the 337 mutants for which the transposon insertion site was determined, very few corresponded to genes that had been identified in the two previous screens conducted on S. pneumoniae[22,23], confirming the lack of overlapping of genes identified by distinct STM work on the same microorganism. Genes were grouped into four classes according to their defects in the four different models of infection tested: the lung alone; the lung and blood; the lung and the naso-pharynx; or all three organs. A number of genes were common to all three tissues, while others revealed the existence of S. pneumoniae tissue-specific virulence factors. Each of the four classes of genes includes transcriptional regulators, suggesting that the tissue-specific regulation is a significant character of the virulence of S. pneumoniae. In addition, a high proportion of genes identified in that study encoded putative proteins of as yet unknown function. It is worth mentioning that only 25 of the 337 putative attenuated candidates have been actually further tested individually for reduced virulence. Thus, the role of the genes inactivated in the other mutants in bacterial virulence remains to be established.

In S. aureus, a total of 237 candidates were selected by STM for defects of growth and survival in at least one of the three different infection models tested (bacteraemia, presence in the wounds or abscesses in the mouse, and in a model of rabbit endocarditis), after screening of 1520 mutants in the mouse [24]. Less than 10% of these candidates were selected in all the models. Eighty-five mutants were characterized genetically. Only 55% of the genes identified could be assigned a functional class after Blast similarity analysis. Six of these mutants were further tested individually in abscesses, wound and LD50 infection models. Three of these mutants contained insertions in homologues of peptide transporter genes, emphasizing the key role of aminoacid – or peptide – transporters for the establishment of the infectious process, i.e., the survival of the bacteria in its niche.

The genes responsible for tissue-specific tropism were also studied by STM in the Yersinia genus. Three species are pathogenic for humans: Y. enterocolitica, Y. pseudotuberculosis and Y. pestis. The two first cause gastro-enteritis, generally following ingestion of contaminated food, and the latter is the causative agent of plague. These three bacterial species possess a very conserved virulence plasmid of about 70 kb (pYV). They also share a high degree of nucleotide identity, in spite of causing very distinct pathologies. Wren and collaborators looked for determinants common to Y. pseudotuberculosis and Y. pestis, necessary for establishment of infection, and specific genes contributing to the particular tropism of Y. pseudotuberculosis. STM was applied to Y. pseudotuberculosis and led to the selection of 31 candidates among 603 mutants tested [25]. More than 30% of the identified genes take part in the production of the cell envelope (synthesis of the LPS), 25% are of unknown function, and the remainder have very different functions. Among the 20 mutants that were tested in vivo, 14 showed a significant attenuation of virulence. By comparing the genome of both organisms, the authors identified five mutants that are affected in genes unique to the genome of Y. pseudotuberculosis. Since Y. pestis colonizes the lung, these genes that are necessary to establish infection could participate to colonization of the intestine, and thus explain the different tropism of the pathogen. One of the mutants is inactivated in a gene, pldA that encodes a phospholipase. This gene is widely distributed in Enterobacteriaceae, suggesting that the corresponding protein might be a general factor implied in the colonization of the gastro-intestinal tract in other enteric bacterial pathogens.

5Mechanisms of survival and persistence within the host

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Signature-tagged mutagenesis
  5. 3Identification by STM of the role of mycobacterial lipids in virulence
  6. 4Host tropism
  7. 5Mechanisms of survival and persistence within the host
  8. 6Crossing of the blood–brain barrier
  9. 7Concluding remarks
  10. Acknowledgements
  11. References

Bacteria have evolved a broad range of processes to survive and multiply in specific hosts, allowing them to adapt to the different types of stress encountered. The fact that many genes identified by STM are related to metabolism, or encode systems for metabolite acquisition, suggests an important role of adaptation to environment in bacterial virulence.

5.1Adaptation to stress

An extracellular bacterial pathogen must be able to survive in a “hostile” environment before and after adhesion to the host cell surface. For example, Vibrio cholerae develops an acid tolerance response (ATR) that enables it to survive at low pH levels. The importance of the ATR in adaptation of V. cholerae to acidic environment in infected hosts was confirmed by STM in a murine model of infection [26]. In that study, 9600 V. cholerae mutants were tested in the infant mouse model. Sequence analysis of 164 attenuated candidates identified 95 different orf, belonging to diverse functional classes. The 95 attenuated strains were further tested in an acid stress tolerance (ATR) assay, designed to mimic in vivo environments. Nine mutants were shown to be reproducibly attenuated for ATR. Three of the nine corresponding genes (gshB, nqrA, nqrF) probably take part in homeostasis of certain ions like Na+ or K+. In the same organism, Chiang and Mekalanos [27] found previously known virulence genes affecting the toxin co-regulated pilus (tcp genes), as well as candidates affected in metabolic pathways. Most of these mutants were affected in purine biosynthesis, which is a particularly growth-limiting nutrient for many pathogenic bacteria [28].

5.2Nutritional deficiencies

Some nutrients are very rare in the host environment, like amino acids or the ions necessary for enzymatic activities.

5.2.1The case of Helicobacter pylori, an extracellular bacterium

Helicobacter pylori colonizes the gastric mucous membrane and causes an acute inflammatory response, which can progress in various states, from superficial gastritis to gastric cancer. A series of 960 STM mutants were applied in successive rounds of gerbil infections [29]. DNA sequencing of 252 non-colonizing H. pylori mutants identified 47 different genes. Fourteen mutants from different functional groups were individually tested for attenuation in vivo. All of them were identified as heavily attenuated in the animal assay. Two mutants were studied in greater detail (inactivating genes hp0169 and hp 0017, respectively). The authors demonstrated that the protein encoded by gene hp0169 had a collagenase activity and was secreted in the external medium. This protein could serve to enable the recovery of amino acids or short peptides via collagen degradation. Another role of this enzyme might be the degradation of the components of immunity such as the IgA antibodies. The gene hp0017 encodes a putative ATPase involved in natural transformation competence of H. pylori. Preliminary evidence suggests that, besides its role in natural transformation, this protein (denoted ComB4) might have an additional, yet unidentified, essential function for the bacterium.

5.2.2Nutritional deficiency of intracellular pathogens

Bacteria that multiply intracellularly are also confronted with nutrient deficiencies. The bacteria of the genus Brucella are facultative intracellular pathogens, able to survive and to multiply in professional and non-professional phagocytes. They have a broad spectrum of mammalian hosts, like mice, cattle or humans, and cause a chronic fever known as Malta fever. The results obtained by STM on various species of Brucella reveal the need for systems to acquire and metabolize the nutrients. For example, B. suis must synthesize many compounds de novo that it does not find in the host cells, like chorismate [30]. Among the 14 different genes identified, genes previously known to be involved in intracellular survival or smooth LPS biosynthesis were found (virB operon genes and manB, respectively). The two other groups of genes included genes possibly involved in gene regulation and genes encoding enzymes involved in metabolic pathways (glucose metabolism and amino acid biosynthesis). In a screen in B. melitensis on 672 mutants, Lestrate et al. [31] Identified 18 genes involved in different functions, including genes involved in nutrient transport, encoding ion transporters, as well as genes involved in the metabolism of DNA and amino acids. In a second STM on 1152 mutants, Lestrate et al. [32] identified 36 new mutants attenuated in vivo. Among the 20 genes having predicted function, half of them are involved in transport or metabolism, confirming that amino acid and sugar metabolism is a major key for in vivo Brucella survival.

5.3Genes involved in colonization processes

Although the genes necessary for colonization are different from one microorganism to another, common characters can be observed, and in particular the mechanisms by which the Gram-negative pathogenic bacteria adhere to their host cells.

5.3.1Involvement of type three secretion systems

TTSS of Gram-negative bacteria intervene in different steps of infection, and play a crucial role in the colonization of the gastro-intestinal tract [33,34]. The importance of TTSS in bacterial virulence was originally reported in the pioneer study of STM [3] in a murine model of the typhoid fever caused by Salmonella. STM was performed on 1152 mutants of S. typhimurium, after intraperitoneal injection. Forty mutants were selected after dot-blot hybrization, and 28 of them were subjected to DNA sequence analysis. Thirteen attenuated candidates corresponded to genes already known for their participation in the virulence process (like spvA or purD); eight were of unknown functions, and five corresponded to transposon insertion into four different genes presenting similarities to genes encoding proteins of the TTSS (and later identified as components of the SPI-2 PAI [35]). The determination of the LD50 of these four mutants confirmed a significant attenuation of virulence. The functional elucidation of individual SPI-2 effectors has generally focused on their association or colocalization with cytoskeletal components or with membranous structures [19]. A key prerequisite to the translocation of all known SPI-2 effectors is secretion of the translocon components SseB, SseC, and SseD, which are found predominantly on the outer surface of the bacterium and are presumed to contact the host membrane (reviewed in [18]).

Other studies indicated that the loss of the TTSS in Y. pseudotuberculosis resulted in a reduction in the capacity of the bacterium to invade the cecum [36] or the intestinal mucous membrane [13]. Mecsas et al. [36] identified effectors (yscHUBL) and a regulator (lcrR) of the TTSS, possibly involved in genes encoding the attachment of Y. pseudotuberculosis to the membrane of the brush border, and/or contributing to survival in the intestinal lumen.

5.3.2Role of pili and fimbriae

In some bacterial species, colonization of the gastro-intestinal (GI) tract requires expression of pili. For example, Chiang and Mekalanos [27] looked for V. cholerae mutants affected in survival, growth and persistence in mouse intestine. Five mutants were isolated, affected in the production of toxin-regulated Pilus, a fimbrial structure required for colonization of the murine host. The other candidates seem to intervene in persistence.

Martindale et al. [37] used STM in the newborn rat model and identified, on a total of 2140 mutants, sixteen candidates affected for colonization of the gastro-intestinal tract in Escherichia coli K1. Several mutants presented insertions in genes related to the anaerobic metabolism, which is a probable consequence of the low oxygen content of the intestine. Genes required for the biosynthesis of the lipopolysaccharide (LPS) were also identified, suggesting that a complete LPS structure is required for survival in the small bowel. Their exact role remains elusive. One insertion occurred in the gene fimH that encodes the tip adhesin on type I pili, filamentous structures that mediate the attachment of bacteria to mannosylated host receptors. The ability of E. coli to cause urinary tract infections is critically dependent on the presence of these pili. However, the role of type I pili on GI colonization remains poorly understood.

The STM study of an uropathogenic E. coli allowed the identification of new genes absent from the non-pathogenic E. coli K-12 strain. Donnenberg and co-workers [38] analyzed 2049 mutants in a murine model of urinary infection, and divided their 19 candidates in four functional groups: (i) type I fimbrial operon; (ii) genes involved in the extra-cellular polysaccharide biosynthesis; (iii) genes of the metabolism; and (iv) genes of unknown function. The mutants presenting the strongest defect belonged to the first two groups, confirming the particular role of type I fimbrial system and extra-cellular polysaccharides in establishing an infection of the urinary tract.

6Crossing of the blood–brain barrier

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Signature-tagged mutagenesis
  5. 3Identification by STM of the role of mycobacterial lipids in virulence
  6. 4Host tropism
  7. 5Mechanisms of survival and persistence within the host
  8. 6Crossing of the blood–brain barrier
  9. 7Concluding remarks
  10. Acknowledgements
  11. References

To reach their site of colonization and/or multiplication, some bacteria must cross the physiological barriers. To cross a barrier, a pathogen must interact with it, resist the host defences, and penetrate across – or in between – the cells constituting the barrier. The most frequently encountered barrier is probably the gastro-intestinal barrier, whose colonization is the first step in the pathogenesis of all enteric and of some systemic infections.

We will not discuss what STM has brought to the understanding of the colonization of the gastro-intestinal tissue (see Section 5.3). West and colleagues published very recently a review on this topic [39]. Instead, we will focus here on the two STM screens that have been undertaken to try to decipher the mechanisms underlying the crossing of the blood–brain barrier (BBB).

The BBB constitutes a particularly impermeable barrier for bacteria. It is thus interesting to understand why only certain bacteria, including E. coli, Neisseria meningitidis, M. tuberculosis, Streptococcus agalactiae, S. pneumoniae and Listeria monocytogenes, are able to cross this barrier and for some of them to be able to invade the brain tissue itself (see for a review [40]). In most cases, invasion of the meninges is a complication of bloodstream invasion, that occurs after primary colonization of the nasopharyngeal tissue in the case of N. meningitidis or the gastrointestinal tissue, in the case of L. monocytogenes (see for reviews [41,42]). Two different structures separate the blood stream from the central nervous system (CNS): the BBB, and the blood–cerebrospinal fluid barrier that is present at the choroid plexuses. The endothelial cells lining the brain capillaries form the BBB. These microvessels are covered by pericytes and outgrowths of astrocytes. Unlike other capillary cells, the endothelial cells of brain capillaries are joined to one another by tight junctions with high electrical resistance (>1000 Ω cm2).

Bacteria enter the CNS following direct interaction with the luminal side of the cerebral endothelial tissue. The fact that only a limited number of bacterial pathogens are able to traverse this barrier suggests that they have developed specific strategies and possess specific attributes.

6.1Gram-positive determinants implicated in brain invasion

Listeria monocytogenes is a Gram-positive bacterium widespread in nature and responsible for sporadic severe infections in humans and other animal species (see [43], and references therein). This organism is a facultative intracellular parasite capable of invading most host cells, including epithelial cells, hepatocytes, fibroblasts, endothelial cells, and macrophages (see [44], for a review). Each step of the intracellular parasitism by L. monocytogenes is dependent upon the production of virulence factors [45]. The major virulence genes identified to date (hly, plcA, plcB, mpl, actA, inlA, and inlB) are clustered into two distinct loci on the chromosome and are controlled by a single pleiotropic regulatory activator, PrfA, which is required for virulence [46]. However, it is most likely that many other genes, involved in various stages of the infection in vivo remain to be discovered.

Listeria monocytogenes has tropism for the brain as well as for the meninges, and clinical syndromes due to L. monocytogenes in CNS are meningitis, meningoencephalitis and abscess formation. L. monocytogenes can use several different mechanisms to invade the CNS. Infection from the blood stream occurs either by direct invasion of endothelial cells by free blood-born bacteria or by bacteria circulating in infected leukocytes. In addition a neural route of infection has been described in which bacteria reach the CNS via intra-axonal transport from peripheral tissues (see for a review [40]).

In order to identify genes possibly involved in passage across the blood–brain barrier, we used STM to identify mutants affected in their multiplication in the brains of infected animals [47]. This study represents the only STM screen performed to date in a Gram-positive pathogen that used the brain as a target organ. We tested a bank of 2000 mutants for their capacity to penetrate and/or to multiply in the brain of infected mice [47]. Forty-one pools of 48 mutants were generated, and each pool was injected into mice at a dose of 106 bacteria per animal to ensure a starting inoculum of approximately 2 × 104 bacteria per mutant in each pool. Sixty putative candidates were identified by dot-blot analysis. However, when tested individually, only eighteen of them appeared to be really attenuated. Determination of the transposon insertion site in these 18 attenuated mutants led to the identification of 10 distinct loci: five loci corresponding to putative cell wall components, and five loci involved proteins participating in various cellular processes. The four mutants that showed the highest LD50 values (>106; i.e., 1.5 log higher than the wild-type strain), all corresponding to genes encoding cell wall components (YtgP, GtcA, PbpX, and Lmo2026) were studied in greater detail. Four identical independent transposon insertions were found in ytgP. This orf encodes a putative integral membrane protein possibly involved in polysaccharide biosynthesis. Of interest, immediately downstream of the inactivated gene, a second orf encodes a homologous protein of identical size. In spite of repeated efforts, we were unable to inactivate this second orf, suggesting that the double mutant might not be viable (unpublished data). The gtcA gene is specifically involved in the glycosylation of teichoic acid (TA) domains. Strikingly, five independent transposon insertions were found in this gene (three of which contained an identical insertion). The fact that integrity of this major cell wall component is required for bacterial virulence suggests that TA might influence the capacity of the bacteria to invade and multiply in the brain, either directly or indirectly by influencing other cell-wall-associated factors involved in virulence. One Tn insertion occurred immediately upstream of a gene encoding a putative penicillin-binding protein sharing 47% identity with PbpX of B. subtilis. PbpX of L. monocytogenes might be involved in cell wall synthesis. The fourth Tn insertion occurred immediately upstream of an orf encoding a 626-residue protein (Lmo2026) of unknown function. The ORF, whose predicted product does not have significant similarity with other proteins in the databases, contains five leucine-rich repeats and a membrane-anchor LPXTG motif in its C-terminal part. Such motifs are generally found in proteins of the internalin family [48]. We are currently testing the possible role of ORF626 in the binding and entry of L. monocytogenes into eucaryotic cells.

The in vivo kinetics of bacterial multiplication of these four mutants (monitored in the spleen and the liver) was similar to that of the wild-type strain until day 3. Then, bacterial counts fell from day 4 and all of the mice fully recovered from the infection. Thus, in the four mutants, attenuation did not result in a defect in the early stage of multiplication in the spleen or in the liver. Moreover, in no case was invasion of the brain completely abolished. These results suggest a defect of persistence in the brain rather than a defect in crossing of the BBB. Direct invasion of the vascular endothelium by bacteria is not the only means of cellular infection. Indeed, experimental data had already demonstrated dissemination of intracellular L. monocytogenes, indicating that phagocyte-facilitated invasion has a role in central nervous system infection [40]. We found no difference in vitro in the intracellular multiplication of the four mutants in mouse macrophages.

The fact that insertions in gtcA and ytgp were repeatedly found suggested the existence of possible hot spots of insertion of the transposon used for mutagenesis. A second STM screen further confirmed this assumption that we performed more recently with the same transposon [49]. In that second screen, we chose the liver as a target organ to ensure a higher number of recovered bacteria after infection (the number of bacteria per mouse can be up to ca. 1000-fold higher in the liver than in the brain [50]). Forty different loci were identified (unpublished data). These included 7 out of the 10 loci that had been identified in our previous screen. Strikingly, as in the first screen, insertions into the genes ytgP and gtcA were the most frequent, with 11 mutants at each locus. This result demonstrated that these two genes, initially involved in survival in the brain, were more generally involved in the overall in vivo proliferation of the bacterium.

6.2Use of cellular models to elucidate the crossing of BBB by Gram-negative bacteria

In addition to the animal model, cellular models, primarily endothelial cells cultures, were used in STM studies. E. coli K1 is the principal cause of meningitis for newborns. 3360 mutants of E. coli K1 were analyzed in an infection model of HBMEC cells (human brain microvascular endothelial cell) [51]. Very few mutants (less than 0.2%) were identified as attenuated in virulence. Interestingly, a mutant disrupted in the traJ gene was found. The TraJ protein is normally implicated in positive gene regulation of genes necessary for conjugation. This does not seem to be related to pathogenesis. Nevertheless, the traJ locus was shown to influence the ability of E. coli K1 to invade HBMEC in vitro and to cross the BBB in vivo [51]. In a subsequent paper, the authors began to elucidate the mechanism by which TraJ participates to virulence. They demonstrated that TraJ contributes to the early systemic dissemination of E. coli K1 in the oral infection process via specific TraJ-dependent bacterial interactions with macrophages [52]. Experiments are in progress to understand this phenomenon. It is worthy to notice that this gene was also identified in an IVET screen conducted by the same team [53].

Many genes were discovered that participate in interactions with endothelial cells, or are required for persistence in the brain. However, no specific gene implicated directly in the crossing of BBB has ever been found, giving clues for a better understanding of this phenomenon. The currently acknowledged hypothesis is that crossing of the BBB is the result of a concerted and synergistic action of many genes: abolition of crossing would probably need inactivation of several of them.

7Concluding remarks

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Signature-tagged mutagenesis
  5. 3Identification by STM of the role of mycobacterial lipids in virulence
  6. 4Host tropism
  7. 5Mechanisms of survival and persistence within the host
  8. 6Crossing of the blood–brain barrier
  9. 7Concluding remarks
  10. Acknowledgements
  11. References

Following the first STM study described in S. typhimurium, STM screens were adapted to a broad range of pathogenic Gram-positive and Gram-negative bacteria (see Table 1), covering a variety of diseases (including enteric or systemic diseases, or infections affecting the respiratory and urinary tracts). The animal models were most frequently employed because they are likely to reflect human infection. In some cases, where no convenient in vivo model was available, in vitro models were developed, as for examples for Brucella species [30], E. coli K1 [51] or N. meningitidis[54].

Studies on Gram-positive bacteria represent only one fifth of the total number of STM studies published to date. However, they were generally more extensive, either because they used a greater number of mutants, or because they were carried out using several models. As expected, the majority of the published STM studies found previously known virulence genes. Several hundreds of new factors have been also identified, among which numerous new genes have not yet been assigned a precise role in virulence.

7.1Limitations and conceptual evolutions

Some experimental restrictions exist, that have already been discussed above (see Section 2.2). But there are also limitations inherent to the method. In particular, STM does not allow the identification of certain categories of genes, like those whose inactivation can be compensated by expression of one or more other genes (this is a limitation of genetic screens in general). The use of mixtures of mutants may in some case, allow the complementation of particular defects. For example, a mutant unable to produce a given secreted factor can use the molecules secreted by the neighbouring bacteria of the infecting pool. Very few overlaps were observed between the different STMs, even when applied to the same bacterium, suggesting that most of the studies are far from being exhaustive. This lack of exhaustivity is due to the method itself that is very difficult to standardize, in particular because of the variability inherent to the animal model used for screening. It is also due the criteria chosen to define an attenuated candidate are quite variable (and in particular the thresholds). In some cases, competitions between wild type and mutants are used to establish attenuation (competition index); while in others, the reduction of LD50 is chosen (which is less sensitive). Moreover, the tests performed often lack of completion. Finally, one source of possible erroneous interpretations may arise from a polar effect of the transposon on the downstream genes. However, in the particular case of operons, most often encoding genes involved in the same function, polar effects might, however, be advantageous, i.e., by broadening the sample size. Therefore, for particularly interesting genes, it is necessary to confirm the data by constructing in-frame chromosomal deletion mutants and by complementation.

STM has proven its greater efficacy over other large-scale genetic methods, for the identification of genes involved in pathogenic processes, in terms of numbers of mutants identified as well as efficiency. In vivo expression technology (IVET) constitutes another genomic-scale approach for identifying genes involved in bacterial pathogenesis. IVET allows the identification of genes whose expression is regulated (generally up) during infection. However, in contrast to STM, where the mutants are directly available for further functional analyses, subsequent gene inactivation must be performed in IVET to evaluate the role of the genes identified in bacterial virulence. The general lack of overlapping results produced by IVET and STM in several different microorganisms highlights the usefulness of developing complementary approaches for exhaustive functional genomic analyses [55].


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Signature-tagged mutagenesis
  5. 3Identification by STM of the role of mycobacterial lipids in virulence
  6. 4Host tropism
  7. 5Mechanisms of survival and persistence within the host
  8. 6Crossing of the blood–brain barrier
  9. 7Concluding remarks
  10. Acknowledgements
  11. References

We thank Vladimir Pelicic for his comments and suggestions. We also thank the referees for their very helpful advises. This work was supported by CNRS, INSERM, Université Paris V, and Université Paris VII. Nicolas Autret received a fellowship from the “Ministère de l'Education Nationale de la Recherche et de la Technologie”.


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Signature-tagged mutagenesis
  5. 3Identification by STM of the role of mycobacterial lipids in virulence
  6. 4Host tropism
  7. 5Mechanisms of survival and persistence within the host
  8. 6Crossing of the blood–brain barrier
  9. 7Concluding remarks
  10. Acknowledgements
  11. References
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