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Keywords:

  • cytosolic protein degradation;
  • enzyme;
  • infection;
  • Salmonella typhimurium

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The cytosolic protein degradation pathway, involving ATP-dependent proteases and ATP-independent peptidases, is important for modulating several cellular responses. The involvement of pathogen-encoded ATP-dependent proteases is well established during infection. However, the roles of ATP-independent peptidases in this process are not well studied. The functional role of Peptidase N (PepN), an ATP-independent enzyme belonging to the M1 family, during systemic infection of mice by Salmonella enterica serovar Typhimurium (Salmonella typhimurium) was investigated. In a systemic model of infection, the number of CFU of S. typhimurium containing a targeted deletion in peptidase NpepN), compared with wild type, was significantly higher in the lymph node and spleen. In addition, S. typhimurium replicated in the thymus and greatly reduced the number of immature CD4+CD8+ thymocytes in a dose- and time-dependent manner. Strains lacking or overexpressing pepN were used to show that the reduction in the number of thymocytes, but not lymph node cells, depends on a critical number of CFU. These findings establish a role for PepN in reducing the in vivo CFU of S. typhimurium during systemic infection. The implications of these results, in the context of the roles of proteases and peptidases, during host–pathogen interactions are discussed.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cytosolic protein degradation plays an important role in the maintenance of cellular homeostasis and is of great significance in various cellular processes. This pathway can be categorized into two distinct stages based on the need for ATP. The proximal ATP-dependent protein unfolding and proteolytic events are followed by ATP-independent processing and degradation of peptides into free amino acids. La endopeptidase (Lon) and Caseinolytic protease (ClpP) are the major ATP-dependent proteases in bacteria, whereas the ATP-independent events are performed by multiple peptidases, which lead to the generation of free amino acids (Chandu & Nandi, 2004; Nandi et al., 2006).

Salmonella typhimurium is a facultative intracellular pathogen that exhibits a wide host range, infecting humans, cattle, mice and chickens. Infection manifests as enterocolitis in humans and cattle; however, in mice, the organism crosses the intestinal barrier and infects systemic organs. The roles of two Salmonella pathogenicity islands (SPIs), each encoding a type III secretion system for virulence, are well studied. SPI1 is involved in infecting intestinal cells whereas SPI2 is responsible for systemic pathogenesis (van Asten & van Dijk, 2005). Recently, there have been several studies that implicate ATP-dependent proteases in the determination of virulence. Salmonella typhimuriumΔlon survives poorly within macrophages, colonizes systemic organs less efficiently and is highly attenuated (Takaya et al., 2003). Furthermore, Lon degrades key proteins and negatively regulates the expression of SPI1 (Boddicker & Jones, 2004; Takaya et al., 2005). Also, S. typhimuriumΔclpP exhibits reduced capacity to survive within macrophages and is impaired in the colonization of the spleen and liver in oral and systemic models of infection (Yamamoto et al., 2001).

Although there is relatively better knowledge on the effects of the ATP-dependent proteases, the roles of ATP-independent peptidases during the infection process are not well established. PepN belongs to the M1 family of metallopeptidases and is responsible for the majority of the cytosolic aminopeptidase activity in Escherichia coli. Unlike most aminopeptidases, it cleaves both amino and endopeptidase substrates; in fact, it is the only known aminoendopeptidase in eubacteria. Furthermore, E. coli lacking pepN is more adept in resisting sodium salicylate-induced stress, demonstrating a role for pepN in modulating selective stress responses (Chandu & Nandi, 2003; Chandu et al., 2003). Interestingly, PepN from Brucella melitensis is recognized by sera from patients with acute and chronic brucellosis, suggesting that it is an immunogenic aminopeptidase with possible diagnostic significance (Contreras-Rodriguez et al., 2003). Notably, pepN is downregulated during infection of a macrophage cell line by S. typhimurium (Eriksson et al., 2003). However, there are no functional data to validate this observation in an infection model system. Therefore, this study was initiated to investigate the functional role of pepN during systemic infection of mice with S. typhimurium.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial strains and culture conditions

Salmonella typhimurium NCTC 12023 (Chakravortty et al., 2005) was used as the wild type (WT) strain for construction of ΔpepN, using the following strategy (Chandu et al., 2003): The E. coli pepN construct harboring the kanamycin resistance cassette in place of conserved catalytic motifs was transformed into S. typhimurium containing pKD46 encoding λRed. Positive clones were selected on Luria–Bertani (LB) agar plates containing 30 μg mL−1 kanamycin at 30 °C and later cured of pKD46 by overnight growth at 42 °C. The pepN overexpression plasmid, #5423, containing the endogenous promoter, was a kind gift from Charles Miller, University of Illinois, Urbana-Champagne. This plasmid was used to generate pepN overexpressing transformants in both WT and ΔpepN (Table 1) that were grown in LB containing 100 μg mL−1 ampicillin. The WT strain STM8c and ΔinvA were gifted by E. Charpentier, University of Vienna, Austria, and have been characterized previously (Galan & Curtiss, 1989). ΔinvA was grown in LB containing 50 μg mL−1 tetracycline.

Table 1. Salmonella typhimurium strains used in this study
Sl. No.Strain designationsStrain characteristics
1WTS. typhimurium NCTC 12023
2ΔpepNWT containing a targeted deletion in pepN
3WT/VectorWT transformed with pBR322
4ΔpepN/VectorΔpepN transformed with pBR322
5WT/pepNWT transformed with #5423, the pepN overexpressing plasmid
6ΔpepN/pepNΔpepN transformed with #5423, the pepN overexpressing plasmid

Strains were grown in LB broth in the presence or absence of appropriate antibiotics. Different strains were streaked on Salmonella Shigella agar (SSA) plates and incubated overnight at 37 °C. A single colony was picked and inoculated in LB broth for 12 h at 37 °C. This preinoculum was diluted in LB broth and cultured for 3 h (log phase) or overnight (static phase) and used for appropriate experiments. Bacteria were centrifuged, washed, resuspended in Hank's-buffered salt solution (HBSS) or phosphate-buffered saline (PBS) and used for infection.

Enzyme assays

Enzyme assays were performed as described previously (Chandu & Nandi, 2003). Briefly, cytosolic lysates (5–25 μg total protein) were incubated with 0.5 mM Arg-AMC, 0.5 mM Suc-LLVY-AMC or 0.5 mM Cbz-GGL-βNA (Sigma Chemical Co., St Louis, MO). Free βNA or AMC released post cleavage was measured and specific activity was calculated as the nanomoles of βNA or AMC released per microgram of protein per hour at 37 °C.

Mice

BALB/c female mice (5–8-week age group) were procured from the Central Animal facility, IISc, or the National Institute of Nutrition, Hyderabad. Mice were housed in the departmental facility according to institutional guidelines.

Isolation of thymocytes and lymph node cells

Mice were infected with indicated doses of CFU of S. typhimurium via the intraperitoneal route. After appropriate days, the mesenteric lymph node and thymus were dissected and the CFU and cell numbers were determined from the same organ. Cells were removed by teasing, washed and an aliquot was used for fluorescence-activated cell sorting (FACS) staining or determination of viable cell numbers using Trypan Blue. Cells were stained with monoclonal antibodies to CD4 and CD8 conjugated to fluorescein isothiocyanate (FITC) and phycoerythrin (PE), respectively (BD, San Diego, CA), and analyzed on a Becton Dickinson FACScan flow cytometer. The cellquest (Becton Dickinson) software was used for acquisition, and winlist (Verity, Topsham, ME) software was used for further data analysis, as described previously (Prasanna & Nandi, 2004).

Macrophage isolation and culture

Mice were injected via the intraperitoneal route with c. 4 mL of 4% Brewer's Thioglycollate broth (Himedia) and sacrificed after 4–5 days. Thioglycollate-elicited macrophages were harvested by peritoneal lavage with c. 5 mL of 0.32 M ice-cold sucrose. Cells were washed, resuspended in appropriate volume of plain Roswell Park Memorial Institute (RPMI) and plated in 96-well flatbottom cell culture plates (Tarsons, India). Cells were incubated for 2 h at 37 °C in a CO2 incubator to facilitate adherence to plastic. At the end of the incubation period, nonadherent cells were removed by washing with HBSS. Cells were detached from the plate using ice cold 0.5 mM EDTA, washed and purity was confirmed by FACS analysis. Viable cells were counted by the Trypan blue exclusion method using a hemocytometer. Macrophages were cultured at 37 °C, in the presence of 5% CO2, in RPMI 1640 medium supplemented with 25 mM HEPES (Sigma), 2 mM l-glutamine (Himedia, India), 5 μM β-mercaptoethanol (E Merck, San Diego, CA), 100 μg mL−1 penicillin, 250 μg mL−1 streptomycin, 50 μg mL−1 gentamicin (Himedia) and 10% heat-inactivated fetal bovine serum (FBS) (Sigma).

Macrophage cytotoxicity assay

The crystal violet staining protocol was standardized to study the macrophage cytotoxicity caused by S. typhimurium (Lundberg et al., 1999). Briefly, adherent thioglycollate-elicited macrophages were infected with log-phase-grown bacteria at different multiplicities of infection (MOI) for a period of 1 h in RPMI 10% FBS without antibiotics. Cells were washed with PBS, fresh medium with antibiotics was added and incubated further for 1 h. After washing with PBS, cells were stained with 100 μL of 0.2% crystal violet in 20% methanol for 15 min at 37 °C. Extensive washings were performed with water to remove excess dye and the plates were dried completely. A solution comprising 0.1 M sodium citrate (pH 5.4) and 20% methanol was added to the wells and incubation was performed for 30 min at 37 °C. At the end of the incubation period, the absorbance was measured at 620 nm. The absorbance of untreated cells was taken as 100%. The percentage of cell survival was defined as the relative absorbance of treated vs. untreated cells.

Intracellular bacterial survival assay

Thioglycollate-elicited peritoneal macrophages were plated at c. 2–5 × 105 cells  per well in RPMI 10% FBS medium in 24-well plates and incubated overnight at 37 °C. The cells were infected with static-phase bacteria with indicated MOI. Synchronization and enhancement of infection was achieved by centrifugation of plates at c. 50 g for 5 min. Phagocytosis was facilitated by incubating cells for a period of 30 min. At the end of the incubation period, extracellular bacteria were removed by washing three times with PBS. Fresh medium containing 100 μg mL−1 gentamicin was added to the wells and incubated further for a period of 1 h to kill the residual extracellular bacteria. After washing with PBS, fresh medium containing 20 μg mL−1 gentamicin was added. The cells were incubated for appropriate durations of time under this condition. At the end of the incubation period, cells were washed with PBS three times and lysis was achieved by incubating the cells with 500 μL of 0.1% Triton X-100 for 10 min at room temperature. The cell lysates thus obtained were appropriately diluted in PBS and were plated in triplicate individually on SSA plates. The colonies were counted after overnight incubation at 37 °C, and CFU per 2–5 × 105 cells were calculated.

Nitrite assay

Thioglycollate-elicited macrophages (c. 2 × 105 cells well−1) were seeded in 96-well flatbottom plates. The cells were treated with lipopolysaccharide (Sigma) or infected at indicated MOI of static-phase cultures of live or heat-killed S. typhimurium (WT or ΔpepN). The cells were incubated for 1 h under an antibiotic free condition. At the end of the incubation period, the cells were washed and a fresh medium with antibiotics was added. The cells were incubated for specified time periods at 37 °C in the CO2 incubator. Culture supernatants were harvested at different time points and nitrite was quantified using the Griess reagent (Malu et al., 2003).

Determination of burden of infection

Mice were infected via the intraperitoneal route with log-phase cultures of indicated CFU of either WT, ΔpepN or pepN overexpression strains. Postinfection, different organs were weighed and lysates were prepared in PBS using the Dounce homogenizer. The lysates were appropriately diluted and plated on SSA. The data were represented as CFU per organ and graphpad.prism version 4 was used to plot values.

Mice survival assays

Mice were infected via the intraperitoneal route with log-phase cultures of WT or ΔpepN. The animals were monitored for death every 12 h, and graphpad.prism version 4 was used to plot survival curves.

Statistical analysis

The statistical significances of the CFU per organ values obtained from infection burden experiments were tested by the Mann–Whitney U-test. The statistical significance of the survival curves was tested by a log rank test using graphpad.prism version 4.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Selected amino and endopeptidase substrates are not cleaved by ΔpepN

The endogenous pepN was replaced with a homologous disrupted pepN harboring the kanamycin resistance cassette to generate ΔpepN. After PCR amplification, genomic DNA from WT amplified a band of c. 2.6 kb, whereas the identical primers amplified a band of c. 3.8 kb from ΔpepN, consistent with expected results (Fig. 1a). Cytosolic lysates from ΔpepN and ΔpepN/vector were unable to cleave Suc-LLVY-AMC, an endopeptidase substrate, and Arg-AMC, an aminopeptidase substrate (Fig. 1b). However, overexpression of pepN enhanced the cleavage of these substrates. Notably, there was no difference in the hydrolysis of Cbz-GGL-βNA, which is not cleaved by pepN. These studies, using ΔpepN and pepN overexpression, demonstrate that pepN from S. typhimurium encodes an enzyme that is an aminoendopeptidase, similar to its counterpart in E. coli (Chandu & Nandi, 2003; Chandu et al., 2003).

image

Figure 1.  PepN, an aminoendopeptidase, does not modulate in vitro growth of Salmonella typhimurium. (a) Genomic DNA from S. typhimurium WT (lane 1) and ΔpepN (lane 2) were amplified using gene-specific primers. Lane M represents the 1 kb ladder. (b) Cytosolic lysates of WT, ΔpepN and pepN overexpressing strains were assayed for their ability to cleave Cbz-GGL-βNA, Suc-LLVY-AMC and Arg-AMC. The data represent mean±SE, n=3 experiments. (c) Indicated strains were grown in LB media at 37°C and cultures were aliquoted at different time points for reading at OD600 nm or were plated on SS agar plates for CFU analysis. The data are representative of two independent experiments.

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Next, we investigated whether PepN modulated the in vitro growth of S. typhimurium. There was no difference in terms of growth and CFU (Fig. 1c) between WT, ΔpepN and pepN overexpressing strains in LB at all the time points studied. Thus, pepN does not play a role in the growth and survival of S. typhimurium in a nutrient-rich medium.

PepN does not modulate macrophage cytotoxicity, intracellular bacterial survival and nitrite production induced by S. typhimurium

After gaining entry into the gastrointestinal tract, S. typhimurium infects host macrophages and induces cell death. This rapid death of macrophages is dependent on SPI1 and host caspase-1, which leads to successful colonization of the intestinal tract. To study the effect of pepN on the quick death of macrophages induced by S. typhimurium, thioglycollate-elicited macrophages were infected at indicated MOI and the extent of death was quantified. Consistent with previous results, ΔinvA was unable to cause the rapid death of macrophages due to an impairment of the inner membrane component of the SPI1 secretion apparatus (Galan & Curtiss, 1989). However, no significant difference was found between WT and ΔpepN (Fig. 2a), which demonstrates that pepN does not play a role in macrophage cytotoxicity caused by S. typhimurium.

image

Figure 2. Salmonella typhimurium-encoded pepN does not modulate macrophage cytoxicity, intracellular bacterial survival and nitrite production. (a) Approximately 105 thioglycollate-elicited macrophages from BALB/c mice were infected with indicated MOI of the above-mentioned strains for 1 h, and macrophage viability was determined. (b) Thioglycollate-elicited macrophages were infected at two different MOI with indicated strains. Macrophages were lysed at indicated periods of time and the lysates were plated. (c) Thioglycollate-elicited macrophages were stimulated with lipopolysaccharide (10 μg mL−1), live or heat-killed strains of S. typhimurium (MOI 1) and incubated for different periods of time. At the end of each incubation period, nitrite production by macrophages was quantified. The data show mean±SD for triplicate determinations. The data are representative of two independent experiments.

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Salmonella typhimurium is a facultative intracellular pathogen that resides and replicates within macrophages. Intracellular survival was investigated at two different MOI using thioglycollate-elicited macrophages. The extent of intracellular survival exhibited by WT and ΔpepN was similar (Fig. 2b). Thus, pepN does not modulate intracellular survival of S. typhimurium within thioglycollate-elicited macrophages.

Macrophages are capable of producing a repertoire of reactive radicals that inhibit or kill intracellular pathogens. Nitric oxide is an important effector molecule and iNos−/− mice are susceptible to systemic salmonellosis (Mastroeni et al., 2000). The ability of live and heat-killed WT and ΔpepN to modulate nitrite production by thioglycollate-elicited macrophages was studied. The culture supernatants were harvested every 24 h and amounts of nitrite were quantified. Macrophages treated with lipopolysaccharide as a positive control induced high amounts of nitrite (Fig. 2c). However, both WT and ΔpepN elicited similar amounts of nitrite production, which demonstrates that pepN does not modulate nitrite production by macrophages.

Role of pepN during a systemic model of infection by S. typhimurium

Mice were infected with 103 CFU of WT or ΔpepN via the intraperitoneal route of infection. After 3 days of infection, the number of CFU for WT and ΔpepN was approximately the same in the mesenteric lymph node and spleen. However, on day 5, ΔpepN exhibited significantly more CFU (c. 18–58-fold) compared with WT (Fig. 3a), which clearly demonstrates that pepN reduced the in vivo growth of S. typhimurium during systemic infection in mice.

image

Figure 3. Salmonella typhimuriumΔpepN demonstrates increased number of CFU during systemic infection. (a) Groups of five BALB/c mice were administered with 103 CFU of WT or ΔpepN via the intraperitoneal route of infection. The mesenteric lymph node and spleen were dissected at indicated intervals of time and the infection burden was determined. Increased number of CFU of ΔpepN was found to be statistically significant at P<0.05. The data are representative of two independent experiments. (b) Groups of five BALB/c mice were administered intraperitoneal with 102, 103 and 104 CFU of WT or ΔpepN and monitored for death. The data are representative of two independent experiments.

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The above result led to the question of whether this burden of infection increased the death of mice. At the highest dose of CFU used (104), all mice died after 5 days of infection, whereas at lower doses of CFU (102 and 103) all mice died by day 7. However, there was no significant difference in the death profile between WT and ΔpepN (Fig. 3b).

Effect of pepN overexpression during the systemic model of infection by S. typhimurium

In order to confirm the role of pepN, studies using pepN overexpressing strains (WT/pepN, ΔpepN/pepN) and their corresponding vector controls (WT/vector and ΔpepN/vector) were designed. Mice were infected intraperitoneal with 103 CFU and the burden of infection in systemic organs was determined on indicated days after infection. As observed in Fig. 4, there was no significant difference in the number of CFU between WT/vector and ΔpepN/vector after 3 days of infection. However, overexpression of pepN reduced CFU after 3 days of infection. After 5 days of infection, the number of CFU of ΔpepN/vector was far greater compared with WT/vector (40–169-fold); however, pepN overexpression greatly reduced the number of CFU. Together, data from ΔpepN and pepN overexpressing strains confirmed that the number of CFU by S. typhimurium during systemic infection of mice is modulated by pepN.

image

Figure 4.  Overexpression of pepN reduces the number of CFU of Salmonella typhimurium during systemic infection. Groups of five BALB/c mice were infected with 103 CFU of indicated strains via the intraperitoneal route. The mesenteric lymph node and spleen were dissected at indicated intervals of time and the infection burden was determined. The difference was found to be statistically significant at P<0.05.

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Effect of S. typhimurium infection on lymph node cells and thymocytes

Infection by several pathogens reduces the number of thymocytes due to increased apoptosis (Savino, 2006); however, the effect of S. typhimurium infection on thymocytes has not been studied. Mice were injected intraperitoneal with 10 or 103 CFU of S. typhimurium and the intracellular bacterial proliferation and cell numbers in the lymph node and thymus were determined. The number of CFU from mesenteric lymph node cells was enhanced after 5 days of infection with 103 CFU compared with 10 CFU (Fig. 5a). Also, there was a slight reduction in the numbers of lymph node cells isolated in mice injected with 103 but not 10 CFU (Fig. 5b). However, the numbers of CD4+ and CD8+ mature T cell subpopulations in infected mice were comparable to uninfected controls (Fig. 5c–e). Concurrently, the number of CFU from the thymus was increased after 5 days of infection with 103 CFU compared with 10 CFU (Fig. 5f). Thymocyte numbers were not greatly affected upon infection with 10 CFU after 3 or 5 days postinfection. However, infection with 103 CFU greatly reduced the number of thymocytes after 5 days of infection but not earlier (day 3) (Fig. 5g). FACS analysis revealed that this reduction in thymocyte numbers occurred in the immature CD4+CD8+ subpopulation (Fig. 5j).

image

Figure 5. Salmonella typhimurium infection reduces CD4+CD8+ thymocytes in a systemic model of infection. BALB/c mice were injected intraperitoneal with c.10 and c.103 CFU of WT, and the infection burden and total cell count of mesenteric lymph node (a, b) and thymus (f, g) were determined after 3 or 5 days postinfection. The data shown are mean±SE of n=3 mice. The data are representative of two independent experiments. Lymph node cells (c–e) and thymocytes (h–j) from uninfected mice or infected with c.10 and c.103 CFU for 5 days were stained with monoclonal antibodies to CD4 and CD8, and FACS analysis was performed.

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PepN modulates the number of thymocytes during S. typhimurium infection

The modulation of CFU in lymph node and spleen by ΔpepN and pepN overexpressing strains led to investigation of the effect on the host response with respect to thymocyte numbers. As observed in Fig. 6a, the number of CFU greatly increased in the lymph node upon infection with the ΔpepN/vector. This increase was greatly reduced upon overexpression of pepN, which is consistent with previous results (Fig. 4). There was a slight reduction in the number of lymph node cells upon infection; however, there was no major difference upon infection with ΔpepN/vector and pepN overexpressing strains (Fig. 6b). Compared with the WT/vector, infection with the ΔpepN/vector greatly increased CFU in the thymus (Fig. 6c). However, this great increase in CFU did not further reduce thymocyte numbers compared with infection with WT/vector. Overexpression of pepN reduced the number of CFU in the thymus, which led to increased numbers of thymocytes (Fig. 6d). This relationship is shown clearly in Fig. 6f where an increased load of CFU (compare ΔpepN and WT) did not lead to further reduction in thymocyte numbers; however, over expression of pepN resulted in lower loads of CFU, which, in turn, increased the number of thymocytes. Importantly, this relationship between CFU and cell numbers was not observed in case of mesenteric lymph node cells (Fig. 6e).

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Figure 6.  Overexpression of pepN decreases the number of CFU and increases thymocyte numbers in mice infected with Salmonella typhimurium. BALB/c mice were infected with c.103 CFU of indicated strains via the intraperitoneal route. The mesenteric lymph node (a, b) and thymus (c, d) were dissected 5 days postinjection and the infection burden and cell count per organ were determined. The data shown are mean±SE of n=3 mice. The correlation between cell number and CFU in the lymph node (e) and thymus (f) is shown.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

An emerging area of host–pathogen interactions is the role of enzymes involved in cytosolic protein degradation. There are, at least, three main mechanisms by which pathogen-encoded enzymes are involved during infection. First, these enzymes may be directly involved in the breakdown of host proteins into amino acids. For example, the enzymes involved in the breakdown of hemoglobin by Plasmodium falciparum play a crucial role (Liu et al., 2006). Second, proteases may degrade regulatory proteins and modify the expression of important proteins required for virulence (Boddicker & Jones, 2004; Takaya et al., 2005). Finally, they may be involved in resisting reactive radicals generated by host defense mechanisms (Darwin et al., 2003). Although the roles of proteases in modulating infection are well known, there are few reports on the functional roles of peptidases during infection. A periodontal pathogen, Porphyromonas gingivalis, lacking dipeptidyl aminopeptidase IV is less virulent compared with WT probably due to reduced destruction of connective tissue (Yagishita et al., 2001; Kumagai et al., 2003). Also, the intra-erythrocytic development of the malaria parasite is blocked by Bestatin, an aminopeptidase inhibitor. Overexpression of a leucyl aminopeptidase by Plasmodium falciparum reduces the efficacy of Bestatin in blocking growth, demonstrating that this aminopeptidase may be one of the targets of Bestatin (Gardiner et al., 2006). Some aminopeptidases from S. typhimurium have been biochemically characterized (Mathew et al., 2000; Larsen et al., 2001); however, their role during infection has not been studied.

In this study, a genetic approach was used to evaluate the functional contribution of S. typhimurium-encoded pepN during systemic infection in mice. Salmonella typhimurium-encoded pepN, similar to its counterpart in E. coli (Chandu & Nandi, 2003), cleaved both aminopeptidase and endopeptidase substrates (Fig. 1b). Initially, WT, ΔpepN and pepN overexpressing strains (WT/pepN and ΔpepN/pepN) were characterized in vitro with respect to growth and survival in LB broth. There was no significant difference among the strains in the above-mentioned parameters (Fig. 1c). PepN is not essential for in vitro growth of S. typhimurium, probably due to the presence of redundant peptidases. In fact, mutations in four peptidases (pepN, pepA, pepB and pepD), but not single peptidase genes, in S. typhimurium result in a significant decrease in cytosolic protein degradation (Yen et al., 1980). Also, pepN did not modulate nitrite production by macrophages (Fig. 2c) or macrophage cytotoxicity caused by S. typhimurium (Fig. 2a). Salmonella typhimurium lacking lon (Takaya et al., 2003) or clpP (Yamamoto et al., 2001) are highly sensitive to oxidative stress compared with the WT and survive less efficiently within macrophages. However, no difference was observed between the ability of WT and ΔpepN to survive within thioglycollate-elicited macrophages (Fig. 2b). Thus, ΔpepN was not compromised in its ability to interact with macrophages under in vitro conditions. Notably, these studies clearly demonstrate that the phenotypes of ATP-dependent proteases, lon and clpP, are distinct from that of pepN, an ATP-independent peptidase involved in cytosolic protein degradation.

There are several evidences to demonstrate an in vivo role for S. typhimurium-encoded pepN during systemic infection of mice. First, 5 days after infection, the number of CFU of ΔpepN was far greater in comparison with WT in the mesenteric lymph node and spleen (Fig. 3). Previous studies have shown that transformation of S. typhimurium with plasmids reduces virulence (Abromaitis et al., 2005; Knodler et al., 2005). However, studies with untransformed or vector transformed strains (Figs 3, 4 and 6) demonstrated the role of PepN in reducing CFU in systemic organs. Overexpression with pepN reduced CFU in the spleen, lymph node and thymus, demonstrating that this effect was pepN-dependent (Figs 4 and 6). Together, the data from ΔpepN and pepN overexpressing strains demonstrate a functional role of pepN in reducing the number of CFU in spleen, lymph node and thymus during systemic infection. Although the number of CFU increased upon infection with ΔpepN compared with WT, there was no difference in the numbers or kinetics of mice survival (Fig. 3b). It is important to note that the increase in the infection burden in case of ΔpepN was observed at a later time point. Hence, it is possible that the high infection burden at a later time point did not translate into reduced survival of mice.

Infection with several pathogens, e.g. Trypanosoma cruzi, Francisella tularensis, mouse hepatitis virus, etc., causes thymic atrophy. The induction of pathogen-induced thymocyte apoptosis is due to an excessive host response due to the production of steroids, chemokines, cytokines, etc. (Savino, 2006). However, there are no studies on the effect of S. typhimurium infection on thymocytes. The important aspects of this part of the study are as follows: first, S. typhimurium replicated in the thymus and reduced the number of thymocytes in a dose- and time-dependent manner (Fig. 5). Therefore, infection with c.10 CFU after 5 days of infection does not lead to significant reduction in thymocytes unlike that observed with c.103 CFU. This reduction in the number of thymocytes occurs later during infection (day 5 compared with day 3). Second, the number of CFU in the thymus was less compared with that observed in the lymph node (Figs 5 and 6). Despite this higher CFU load in the lymph node, there was no major reduction in cellular subpopulations. However, replication of S. typhimurium in the thymus greatly reduced the number of immature CD4+CD8+ thymocytes, as shown previously with other pathogens (Savino, 2006). Third, WT/vector, ΔpepN/vector and pepN overexpressing strains were used to closely study the relationship between the number of CFU and thymocyte numbers during S. typhimurium infection (Fig. 6). It appears that beyond a certain threshold limit, increased CFU (compare WT/vector and ΔpepN/vector) does not translate into an increased reduction in thymocyte numbers. However, reduction in CFU (by overexpressing pepN) below this threshold limit increased the number of thymocytes during S. typhimurium infection. Importantly, this relationship between the load of CFU and host immune cell numbers was observed for immature CD4+CD8+ thymocytes but not mature CD4+ or CD8+ lymph node T cells (Fig. 6). Additional studies are required to investigate the mechanisms responsible for the reduction in thymocytes during infection with S. typhimurium.

PepN and its orthologs possess specialized functions in organisms from different kingdoms. These roles can be broadly categorized into two distinct groups. In the first group, pepN and its orthologs are required for some functions. For example, Lactobacillis lactis lacking pepN displays lowered growth in medium containing casein as a carbon source (Mierau et al., 1996). Further, mice lacking puromycin-sensitive aminopeptidase are sterile due to defective reproductive processes (Osada et al., 2001a, b). Also, mice lacking ERAAP1 display unstable MHC class I molecules on the surface and lowered CD8+ T cell responses (Hammer et al., 2006). In the second group, the absence of pepN and its orthologs enables an organism to resist and grow better under some stress conditions. For example, when glucose is exhausted during diauxic growth, glycogen accumulation occurs in Saccharomyces cerevisiae. This accumulation of glycogen is considered to be a marker for stress in yeast but Saccharomyces cerevisiae lacking aap-1 accumulates less glycogen (Caprioglio et al., 1993). Also, E. coli lacking pepN are able to grow better, compared with WT, in the presence of sodium salicylate and overexpression of pepN reverses this phenotype (Chandu & Nandi, 2003). It appears that pepN encoded by S. typhimurium belongs to this second group as it is important for restricting the number of CFU at a later stage during systemic infection. There are at least two broad reasons for this effect. First, pepN may reduce the proliferation of S. typhimurium during systemic infection so as to minimize the damage to the host. It is important to point out that some pathogen-encoded genes modulate the number of bacteria to allow for a successful infection that retains the host niche (Tierrez & Garcia-del Portillo, 2005). Alternately, S. typhimurium is exposed to different stress during infection (Rychlik & Barrow, 2005) and pepN may modulate this in vivo stress response. Consequently, ΔpepN may be able to resist increased stress occurring during the later phase of systemic infection, resulting in higher CFU compared with WT. Further work is required to fully comprehend the mechanisms by which pepN modulates the systemic infection of mice by S. typhimurium. In addition, the role of pepN during the oral route of infection, which is more physiologically important, by S. typhimurium needs to be investigated.

This study demonstrates a specialized role for pepN during systemic infection by S. typhimurium in mice. It is important because it documents a functional role for an ATP-independent enzyme involved in cytosolic protein degradation. This study has made a beginning and further investigations are required to understand the mechanisms involved in the process. A comprehensive understanding of the roles of ATP-dependent and ATP-independent enzymes involved in cytosolic degradation will add a novel dimension towards understanding host–pathogen interactions.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors appreciate the encouragement and suggestions by Drs Dipshika Chakravortty, N.V. Joshi, P. Sadhale, R. Manjunath, K. Balaji and members of the DpN laboratory. The authors thank Drs C. Miller and E. Charpentier for the gift of plasmids and strains. The authors are grateful to the staff of the Central Animal Facility and the FACS facility in IISc for their cooperation. This study was funded by grants from the Council of Scientific and Industrial Research and the Department of Biotechnology, Government of India.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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