SEARCH

SEARCH BY CITATION

Keywords:

  • antimicrobial genes;
  • Caenorhabditis elegans;
  • innate immune responses;
  • sequential infection

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Caenorhabditis elegans can be used to study the dynamics of polymicrobial infections, specifically those between Gram-positive and Gram-negative bacteria. With C. elegans, Proteus mirabilis acts as an opportunistic pathogen and does not kill this host. Hence, in the present study, C. elegans was immunochallenged by pre-infecting it with the pathogen Staphylococcus aureus in order to study the subsequent effect of P. mirabilis on the host. It was found that 12 hrs of S. aureus and 80 hrs of subsequent P. mirabilis infection significantly reduced the life span of exposed C. elegans by 80%. However, preinfection with S. aureus for 8 and 4 hrs reduced the life span of C. elegans by only 60 and 30%, respectively. Further, there was greater production of reactive oxygen species in the sequentially infected samples than in the S. aureus and P. mirabilis controls. Real time PCR analysis indicated regulation of candidate immune regulatory genes, lysozyme (lys-7), CUB-like proteins (F08G5.6), neuropeptide-like factors (nlp-29), transcription factors of mitogen-activated protein kinase (ATF-7) and daf-2–daf-16 (daf-16), insulin-like signaling pathways and C-type lectin (clec-60 and clec-87) family members during S. aureus and subsequent P. mirabilis-mediated infections, indicating possible roles of, and contributions by, the above factors during host immune responses against these sequential infections. The present findings demonstrate that S. aureus infections increase the vulnerability of the C. elegans host by subverting its immune system, which then permits the opportunistic pathogen P. mirabilis to be pathogenic to this host

List of Abbreviations: 
AMP

antimicrobial peptide

ATF

activating transcription factor

C. elegans

Caenorhabditis elegans

CFU

colony forming unit

DCF

2′, 7′-dichlorfluorescein

DCFH-DA

2′, 7′-dichlorfluorescein-diacetate

E.coli

Escherichia coli

H2O2

hydrogen peroxide

LB medium

Luria Bertani medium

MAPK

mitogen-activated protein kinase

M. nematophilum

Microbacterium nematophilum

NGM

nematode growth medium

NLP

neuropeptide-like protein

O.D.

optical density

P. mirabilis

Proteus mirabilis

ROS

reactive oxygen species

S. aureus

Staphylococcus aureus

Humans are often co-infected or colonized naturally with multiple pathogens, the interactions of which may determine the virulence potential of any one or more of the organisms (1, 2). Despite the abundance of polymicrobial encounters within nature, there are few in vivo models for exploring the biological and pathological systems of interacting species. The soil-dwelling nematode, C. elegans, has been successfully used as an alternative host for the study of host–pathogen interactions (3). It is a newly described, readily available and cheap model organism for studying the pathogenesis of a variety of Gram-negative bacteria, such as Pseudomonas aeruginosa (4–6) and Salmonella enterica serovar Typhimurium (7–9), and Gram-positive bacteria, such as Enterococcus faecalis, Streptococcus pneumonia (10) and S. aureus (11). Peleg et al. demonstrated that C. elegans can be used to study the dynamics of polymicrobial infections (12).

According to Hodgkin et al. the genetically amenable nematode, C. elegans, is ideally suited to identification of several important host factors (13). However, C. elegans lacks a circulatory system and known professional immune cells. The principal site of interaction with most ingested pathogenic microbes is the gut lumen, which is composed of 20 non-renewable intestinal epithelial cells. Recently, researchers have been actively pursuing identification and characterization of components of the C. elegans immune response, in part by monitoring changes in gene expression in response to infection of the intestinal cells (14–17). It is widely reported that C. elegans produces an array of antimicrobial proteins as a part of its immune response to bacterial infections.

Staphylococcus aureus can cause a wide range of illnesses from minor skin infections, such as pimples, impetigo, boils (furuncles), cellulitis folliculitis, carbuncles, scalded skin syndrome and abscesses, to life-threatening diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome, bacteremia and sepsis (18). Infection with this organism most frequently affects the skin, soft tissue, respiratory system, bones, joints, endovascular tissues and wounds.

Staphylococcal infections are extremely difficult to eradicate due to the remarkable capacity of these bacteria to adapt to different environmental conditions both inside and outside of the host organism. Accordingly, in the present investigation, we immunochallenged a host, C. elegans, by preinfecting it with S. aureus with the aim of investigating whether short-term exposure to an infectious pathogen like S. aureus makes this host more vulnerable to subsequently infecting opportunistic pathogens.

Proteus mirabilis, an opportunistic pathogen, is often present in soil, water and the intestinal tracts of many mammals, including humans (19). It can cause blood stream, wound and urinary tract infections in hospitalized individuals (20). In humans, this organism mostly inhabits the urinary tract, where they cause mild infections that are associated with formation of renal and bladder calculi, also known as bladder stones. Although this organism rarely causes disease in healthy hosts, it can do so in individuals undergoing long-term catheterization and those with structural abnormalities of the urinary tract or compromised immune systems.

Caenorhabditis elegans has been used as a versatile model for inducing polymicrobial infections to study host-pathogen interactions (12). P. mirabilis, an opportunistic pathogen that normally affects only patients with underlying disease or compromised immune systems, does not kill C. elegans. So far, it has been unclear whether S. aureus infection induces a sufficiently immunochallenged state in a host to permit infection by an opportunistic bacterium. Our findings demonstrate that S. aureus infection increases vulnerability of the host C. elegans by subverting its immune system, which can result in P. mirabilis being pathogenic to this host.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Bacterial strains and growth conditions

Penicillin resistant Staphylococcus aureus (No.11632) and Proteus mirabilis (No. 7002) were obtained from the American Type Culture Collection, Manassas, VA, USA. E. coli OP50 was provided by the Caenorhabditis Genetics Centre, Minneapolis, MN, USA. All bacterial strains were grown at 37°C and maintained in LB medium.

Caenorhabditis elegans maintenance

Caenorhabditis elegans wild-type strain N2 was maintained at 20°C on NGM (HiMedia, Mumbai, India) with E. coli OP50 as a food source. The animals were age-synchronized by bleaching with commercial bleach and 5M KOH. Stage-synchronized young adult animals were used for different assays.

Caenorhabditis elegans short-time exposure assays

In these assays, young adult C. elegans were exposed to 100% inoculum (32 × 106 cells/mL of LB medium or 0.5 O.D. of cells in 660 nm) of S. aureus for various time intervals (2, 4, 8 and 12 hrs), washed with M9 buffer to remove the bacterial cells from the outer surfaces of the worms and transferred to either E. coli OP50 seeded or unseeded NGM plates to monitor their behavioral changes. This short time exposure assay was essential to the subsequent preinfection experiments.

Detection of bacterial accumulation in nematodes

Bacterial accumulation assays (21) were performed to determine the CFU count of S. aureus inside the exposed worms’ guts after preinfection. Infected worms were washed three times with M9 buffer containing 1 mM sodium azide to inhibit expulsion of bacteria from the worm intestine. Approximately 10 worms were transferred to a 2 mL micro centrifuge tube and the volume made up to 250 μL with M9 buffer. Approximately 400 mg of 1.0 mm silicon carbide particles (Himedia, Mumbai, India) were added to each tube and vortexed at maximum speed for a minute, which completely disrupts the worms but does not affect bacterial survival, and the resulting suspension was diluted and plated onto Aureus agar (Himedia) to determine the CFU count.

Worm killing assay

Approximately twenty age-synchronized young adult hermaphrodite C. elegans were transferred from a lawn of E. coli OP50 to a 48-well culture plate containing the pathogen S. aureus in a liquid medium for preinfection. After preinfection, they were washed and transferred in a liquid medium of P. mirabilis (20% inoculum [0.1 O.D of cells in 660 nm] i.e., 9 × 106 cells/ml of LB medium). C. elegans in food source E. coli OP50 served as control. The plates were incubated at 20°C and monitored every 2 hrs. Animals were considered dead when they showed no response to touch on the solid NGM plates or no pharyngeal contraction was visible.

Reactive oxygen species assay

Reactive oxygen species were measured as previously described (22) with minor modifications. Exposed C.elegans were crushed in ice with a mortar and pestle. Cell lysates were removed by centrifugation and the supernatants used for assay. Briefly, DCFH-DA was dissolved in Tris-Cl to a final concentration of 5 mM before use. For measurement of ROS, the supernatants were incubated with 5 mM DCFH-DA at room temperature for 45 mins. Fluorescence was monitored using a microplate fluorometer using wavelengths of 485 and 530 nm for excitation and emission, respectively. ROS production was calculated using a DCF standard.

Hydrogen peroxide assay

Hydrogen peroxide concentrations were measured by the Wolff method (23). Briefly, worm homogenates were centrifuged at 67 g for 5 mins. 0.1 mL of worm supernatant was added to 0.9 ml of FOX1 reagent (100 μM xylenol orange, 25 mM H2SO4, 250 μM ammonium ferrous sulfate and 100 mM sorbitol) and incubated at room temperature for 30 mins. The reaction mixture was then centrifuged at 67 g for 2 min and the absorbance of the supernatant read at 560 nm. H2O2 production was calculated based on the molar extinction coefficient at 2.24 × 10−5 M−1 CM−1. Results were expressed as generation of nmol H2O2/mg protein.

Total RNA isolation and reverse transcription polymerase chain reaction

To kinetically study the innate immune response of C. elegans against sequential infections, total RNA was isolated from C. elegans that had been infected with S. aureus and subsequently with P. mirabilis (S. aureus 4 hrs, P. mirabilis 20 hrs; S. aureus 4 hrs, P. mirabilis 60 hrs; S. aureus 8 hrs, P. mirabilis 20 hrs; S. aureus 12 hrs, P. mirabilis 20 hrs) along with individually added OP50, S. aureus and P. mirabilis controls. The animals were washed with ice-cold M9 buffer and total RNA isolated using a guanidine thiocyanate/phenol extraction method (24). Total RNA was converted into cDNA using a Superscript III kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instruction.

Real time polymerase chain reaction analysis

Quantitative PCR was performed by analyzing samples taken during the linear range of amplifications. For qPCR analysis, the degrees of expression of seven candidate antimicrobial genes (NLP-29, F08G5.6, Daf-16, ATF-7, lys-7, clec-60 and clec-87) and two unrelated peptide genes (Clk-2, Lbp-5) were analyzed. The primers used in qPCR analysis are shown in Table 1. Real Time PCR was performed with Applied Biosystems equipment (Foster, CA, USA) using Power SYBR Green PCR Master Mix. A negative control without cDNA template was run with every assay to assess the overall specificity and data were normalized relative to C. elegansβ-actin. Final quantitation was achieved by constructing a relative standard curve.

Table 1.  List of primers used in real time PCR analysis
GenePrimer sequence
NLP-29 F: 5′-CGCACAATGGGGATATGG-3′
 R: 5′-CTTTCCCCATCCTCCATACA-3′
F08G5.6 F: 5′-ACCATTGTGGCAAAGGAGAG-3′
 R: 5′-TTGAGCTTCTCGAAAACGTG-3′
Daf-16 F: 5′-AGG AATCCACGGCGTACA-3′
 R: 5′-GAGCCCATCAATGCTCTCTC-3′
ATF-7 F: 5′-CGGAACAATGTC GGATCTTT-3′
 R: 5′-CATGTCGAGTACCGCGTTTA-3′
Lys-7 F: 5′-TTGCAGTACTCTGCCATTCG-3′
 R: 5′-GCACAATAACCCGCTTGTTT-3′
Clec-60 F: 5′-TGTCTGCATTCTTCCAGTCG-3′
 R: 5′-CCCATACCC AGACACCTTTG-3′
Clec-87 F: 5′-AATTCGTGT TCAAGCCAAGG-3′
 R: 5′-AGCCAGTTGATT TTGGTTGG-3′
Clk-2 F: 5′-CCGAATCCTCCATCTTCAAA-3′
 R: 5′-CAATTGTTGCGCCATTACAC-3′
Lbp-5 F: 5′-TCGACGAGACAACACCAGAC-3′
 R: 5′-CACGAATATAGGCACGACGA-3′
β-actin F: 5′-ATCGTCCTCGACTCTGGAGATG-3′
 R: 5′-TCACGTCCAGCCAAGTCAAG-3′

Statistical analysis

All experiments were conducted thrice and one way analysis of variance (using SPSS Ver. 10.00) used to compare the mean values of each treatment. The significance of differences between the means of variables was calculated by using Dunnett's test (P < 0.05). All bacterial infection studies using both solid and liquid assays were performed on at least three separate occasions with comparable results. Data from representative sets of experiments are presented.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Minimum time required for colonization

To determine the minimum time required for preinfection, short time exposure assays were performed. In the presence of 100% inoculum of S. aureus (0.5 O.D of cells in 660 nm), up to 4 hrs of infection induced no differences in the lifespans of infected nematodes when transferred to food source seeded plates immediately after infection. However, compared with exposed animals with normal food sources, infected animals deprived of a food source showed reductions in production of eggs and generation of progeny. The animals’ mortality was significantly increased after 8 hrs of S. aureus preinfection. Hence, for subsequent experiments, 8 hrs of exposure was used as the optimum for preinfections.

Staphylococcus aureus colonization of nematodes’ intestines

To assess the presence and multiplication of pathogenic bacterial strains inside exposed C. elegans’ intestines, a subset of exposed animals were used to evaluate bacterial accumulation inside their guts. CFU counts in the nematodes after the exposure to S. aureus for 4, 8 and 12 hrs were ∼1.1 × 103, ∼5.3 × 103 and ∼54 × 103 cells per worm, respectively. Thus, the number of S. aureus CFU within the C. elegans’ guts increased with more prolonged exposure to the bacteria (Fig. 1). The presence of these significant numbers of S. aureus inside the nematodes’ intestines may have led to persistent infection with increased exposure times. However, no significant numbers of live bacteria were observed inside the animals when they were fed with E. coli OP50 immediately after exposure to infection (i.e., up to 12 hrs).

image

Figure 1. CFU assay. Bar graph showing that the number of S. aureus CFU within the C. elegans gut increased with longer exposure.

Download figure to PowerPoint

Killing of Caenorhabditis elegans by Staphylococcus aureus and Proteus mirabilis

Young adult N2 animals were preinfected with S. aureus for either 4, 8 or 12 hrs, after which the animals were washed with M9 and infected with P. mirabilis for up to 80 hrs. The worms exposed to 4 hrs S. aureus and 80 hrs subsequent P. mirabilis infection had only a 30% mortality and those exposed to S. aureus and P. mirabilis heat-killed 80 hrs only a 5% mortality (Fig. 2). Worms exposed to 8 and 12 hrs preinfection with S. aureus and then 80 hrs P. mirabilis had ∼60 and 80% mortality, respectively. However, worms exposed to 8 hrs and 12 hrs preinfection with S. aureus and then to heat-killed P. mirabilis for 80 hrs had mortalities of 40 and 60%, respectively. No significant mortality was observed in the corresponding P. mirabilis controls (Fig. 3). These results indicate that, compared with OP50, the percentage survival of C. elegans during sequential infections with S. aureus and P. mirabilis was significantly lower (P ≤ 0.05) than was that of the P. mirabilis and S. aureus preinfected OP50 control worms.

image

Figure 2. Assay of killing of C. elegans with sequential S. aureus and P. mirabilis infections.C. elegans was exposed to P. mirabilis, E. coli OP50 and OP50 preinfected with S. aureus served as controls. The worms were analyzed using an inverted fluorescent microscope (Nikon Ti-S, Nikon, Tokyo, Japan). The figure represents data from three independent experiments, each using at least 20 worms. PM, P. mirabilis; SA, S. aureus.

Download figure to PowerPoint

image

Figure 3. Photographs of representative C. elegans after exposure to (a) OP50, (b) P. mirabilis, (c) S. aureus 4 hrs and P. mirabilis 80 hrs, (d) S. aureus 4 hrs and P. mirabilis heat killed 80 hrs (e) S. aureus 8 hrs and P. mirabilis 80 hrs (f) S. aureus 8 hrs and P. mirabilis heat killed 80 hrs (g) S. aureus 12 hrs and P. mirabilis 80 hrs or (h) S. aureus 12 hrs and P. mirabilis heat killed 80 hrs were appraised visually. Representative animals are shown for each of the above listed experimental conditions.

Download figure to PowerPoint

Oxidative stress during sequential infections

To study oxidative stress during sequential infections, overall ROS production and H2O2 production were monitored. Only 8 hr preinfected worms were used for the ROS and H2O2 assays. The sequentially infected worms showed greater ROS and H2O2 production than did the respective S. aureus and P. mirabilis controls (Fig. 4). Even though production of ROS may be involved in host immune responses against invading pathogens, the resultant continuous stress can lead to host damage (25). In the present study, increased production of ROS during sequential infections did lead to host tissue destruction.

image

Figure 4. ROS production during sequential infections (a) Bar graph showing increased production of ROS during sequential infections compared with controls (b) Bar graph showing increased production of H2O2 in sequentially infected samples compared with controls. PM, P. mirabilis; SA, S. aureus.

Download figure to PowerPoint

Degree of expression of candidate antimicrobial genes of different immune pathways

To investigate C. elegans’ antimicrobial response against sequential infections, representative genes of different immune pathways were selected and amplified to assess the amounts of their mRNA. The following selected genes were analyzed: lysozyme family (lys-7), CUB-like proteins (F08G5.6), C-type lectins (clec-60 and clec-87), neuropeptide-like factors (NLP-29) and transcription factors of MAPK (ATF-7) and daf-2–daf-16 (Daf-16) insulin-like signaling pathways.

The C. elegans genome contains numerous candidate antimicrobial genes, including homologs with established roles in host defense. These include 10 homologs of lysozyme (lys-1 to lys-10) (26). Of the 10 lysozyme genes, the degree of expression of lys-7 was selected for study because S. aureus specifically induces expression of lys-7 (26). In the present study, expression of lys-7 mRNA was analyzed kinetically for worms infected sequentially as follows: S. aureus 4 hrs, P. mirabilis 20 hrs; S. aureus 4 hrs, P. mirabilis 60 hrs; S. aureus 8 hrs, P. mirabilis 20 hrs; and S. aureus 12 hrs, P. mirabilis 20 hrs and compared with the following controls: S. aureus 4, 8 and 12 hrs and P. mirabilis 20 and 60 hrs. The sequentially infected samples showed down regulation (Fig. 5a, b) of this gene. These findings suggest that, after S. aureus preinfection, the host had impaired immune defense against the new invading opportunistic pathogen. The F08G5.6 gene belongs to the family of CUB-like proteins. The CUB domain, named for its founding members C1r/C1s, Uegf and Bmp1, contains 110 amino acids that are found in various extracellular and plasma membrane-associated proteins involved in a variety of different functions, including complement activation, development, tissue repair, tumor suppression, and inflammation (27). Previous reports have supported the role of proteins carrying CUB-like domains in C. elegans immunity against several pathogens including M. nematophilum (14), Serratia marcescens (29), and Pseudomonas aeruginosa (15, 24, 26). Of 50 C. elegans genes encoding CUB like factors, F08G5.6 was analyzed in this study; this gene is regulated by the daf-2/daf-16 (30) and p38 MAPK pathways (31). The findings indicated that this gene was upregulated only in S. aureus controls and downregulated in both the sequentially infected samples and P. mirabilis controls (Fig. 5a, c). These findings show that F08G5.6 is specifically induced by S. aureus-mediated infections.

image

Figure 5. Differential gene regulation in sequentially infected samples and their controls. (a) P. mirabilis control indicating degree of expression of candidate antimicrobial genes and unrelated peptides. (b) Lys-7 mRNA is downregulated during sequential infection with S. aureus and P. mirabilis. (c) F08G5.6 mRNA is downregulated during sequential infection with S. aureus and P. mirabilis. (d) ATF-7 mRNA is downregulated during sequential infection with S. aureus and P. mirabilis. P.M, P. mirabilis; S.A, S. aureus.

Download figure to PowerPoint

Caenorhabditis elegans ATF-7, a member of the conserved cyclic AMP–responsive element binding/ATF family, has a pivotal role in regulation of PMK-1 p38 MAPK pathway mediated innate immunity (32). In the present study, expression of ATF-7 mRNA was analyzed kinetically for sequentially infected samples and compared with their respective controls. It was found that the sequentially infected samples showed down regulation of transcription factor ATF-7 (Figs. 5a, d). A similar trend was observed for amounts of daf-16 mRNA, the transcription factor of another immune pathway. daf-16 mRNA encodes the sole C. elegans forkhead box O homolog and functions as a transcription factor that acts in the insulin/insulin-like growth factor-1-mediated signaling pathway that regulates dauer formation, longevity, fat metabolism, stress responses and innate immunity. It regulates these various processes through isoform-specific expression, isoform-specific regulation by different AKT kinases, and differential regulation of target genes (32). Here also, the S. aureus (4, 8 and 12 hrs) and P. mirabilis (20 and 60 hrs) controls showed upregulation of daf-16 whereas the sequentially infected samples showed down regulation of transcription factor daf-16 (Figs. 5a, 6a). These results collectively suggest that during sequential infections, the host cannot withstand the newly invading pathogen. Failure to produce antimicrobial peptides because of down regulation of these transcription factors leads to the host succumbing to the pathogens.

image

Figure 6. Differential gene regulation of sequentially infected samples and their controls. (a) Daf-16 mRNA is downregulated during sequential infection with S. aureus and P. mirabilis. (b) Nlp-29 mRNA is upregulated during sequential infection with S. aureus and P. mirabilis. (c) Clec-60 mRNA is upregulated during sequential infection with S. aureus and P. mirabilis. (d) Clec-87 mRNA is downregulated during sequential infection with S. aureus and P. mirabilis. P.M, P. mirabilis; S.A, S. aureus.

Download figure to PowerPoint

A neuropeptide-like protein gene, nlp-29, that is expressed in the nematode hypodermis and intestine is thought to encode an antimicrobial peptide that is induced in response to both infection with pathogens and physical wounding of worm hypodermal tissue (33, 34). During sequential infections, nlp-29 showed upregulation compared with controls (Figs. 5a, 6b). The amount of nlp-29 transcripts was up to fourfold-higher in sequentially exposed pathogens than in those exposed to either S. aureus or P. mirabilis alone (controls). Hence, it appears that the greater induction of nlp-29 that is a component of the C. elegans innate immune response is directed against sequential infection of the nematode by two different pathogens (Fig. 6b).

C-type lectins reportedly act as pathogen-recognition molecules in the nematode C. elegans, similarly to their role in pathogen-associated molecular pattern recognition in Drosophila (35). Because C. elegans has no cellular immune system, C-type lectins could function as secreted proteins in the intestine and on environmentally exposed surfaces. In the present study, C. elegans C-type lectin gene clec-60 expression was kinetically examined after sequential infection with both S. aureus and P. mirabilis. The qPCR results suggested that clec-60 shows upregulation in both sequentially infected samples and their respective controls (Figs. 5a, 6c). These results indicate that clec-60 might actively participate in S. aureus and P. mirabilis recognition during these exposures.

Another C-type lectin gene, clec-87, showed gradual upregulation in the S. aureus control but it was down regulated in sequentially infected samples and the P. mirabilis control (Figs. 5a, 6d). These results indicate that clec-87 actively participates in S. aureus recognition (Fig. 6d) but does not participate, or participates less actively, in recognition of P. mirabilis.

To study the degree of mRNA expression of unrelated peptides during sequential infections, the clk-2 and lbp-5 genes were analyzed. The clk-2 gene encodes an ortholog of the Saccharomyces cerevisiae telomere length-regulating protein Tel2p. In C. elegans, clk-2 activity is required for DNA damage and S phase replication checkpoints, embryonic development and normal biological rhythms and life span (36). Lbp-5 encodes a predicted intracellular fatty acid binding protein that is most similar to vertebrate muscle. It is predicted to function as an intracellular transporter for small hydrophobic molecules such as lipids and steroid hormones and is mainly required for movement (37). clk-2 and lbp-5 show similar degrees of expression in sequentially infected samples and their respective controls (Fig. 7). These results clearly indicate that unrelated genes do not show any significant regulation during sequential infections.

image

Figure 7. Expression pattern of unrelated peptides during sequential infections. (a) Real-time PCR analysis of Clk-2 mRNA. (b) Real-time PCR analysis of lbp-5 mRNA. PM, P. mirabilis; SA, S. aureus.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Caenorhabditis elegans can be used to study and gain an understanding of the dynamics of polymicrobial infections (12). The present study revealed the impact of an opportunistic pathogen during sequential infection. Unless C. elegans are pre-infected with S. aureus, the opportunistic pathogen P. mirabilis is not lethal to C. elegans. In order to understand the impact of this ‘opportunism’ by selective opportunistic human pathogens, in our study we pre-infected C. elegans with a pathogen that is believed to modify these animals immune system. The main indicator that such modification occurs is the regulation (both up and down) of antimicrobial genes/players during the initial phases of pathogenic infections. However, these regulatory players appear to be transient in nature and eventually persistent infection with the invading pathogens subverts regulation by them. When the immune system has not been challenged, the opportunistic pathogens are unable to produce harmful effects. To understand how exposure to pathogenic bacteria makes a host vulnerable to subsequent infections, and to analyze C. elegans’ specific immune responses against sequential infection with S. aureus and P. mirabilis, we studied the regulation of representative antimicrobial genes.

Sifiri et al tested the ability of 23 S. aureus clinical isolates to kill nematodes in a solid assay (11). A majority (70%) killed < 70% of the nematodes during the course of a standard experiment. Hence, the we used preinfection with S. aureus to immunochallenge these hosts. In the present study, we performed preliminary short time exposure and CFU assays to determine the time required to adequately preinfect C. elegans with S. aureus (Fig. 1). The sequential infection assays revealed that 4 hrs of preinfection with S. aureus, followed by exposure to P. mirabilis for 80 hrs resulted in a mortality of ∼30%. The samples that were preinfected for 8 and 12 hrs showed 60% and 80% mortality, whereas 4, 8 and 12 hrs of S. aureus preinfection followed by exposure of the worms to heat-killed P. mirabilis for 80 hrs produced only about 5, 40 and 60% mortality, respectively. These findings clearly suggest that P. mirabilis is more pathogenic in immunochallenged hosts (Figs. 2 and 3). The dead animals completely lost their body structure and looked like ‘straight sticks’; in some animals we also observed internal hatching (Fig. 3c, e, g). To test for ROS production by C. elegans in response to sequential infections, we performed total ROS production and H2O2 assays. The sequentially infected samples showed increased production of ROS and H2O2 compared with that of the corresponding S. aureus and P. mirabilis controls (Fig. 4). ROS are relatively nonspecific in their capacity to kill cells. They affect invading micro-organisms, but can also cause host tissue damage (25). They may eventually lead to oxidative stress and cause host death in concert with other factors.

Worms have an innate immune system that constitutively expresses certain AMPs, encounters with different pathogens inducing complex mixtures of AMPs (38, 39). After exposure to pathogens, the nematode normally elicits a immune response against the invading microbes. Lysozymes play a very important role in innate immunity and are regulated by Daf-16 and dbl-1/transforming growth factor-β pathways (26). Other studies have been shown that expression of lys-7 is upregulated during infection by other microbes such as M. nematophilum (29) and Vibrio alginolyticus (21). In the present study, compared with the controls (S. aureus[4, 8 and 12 hrs] and P. mirabilis[20 and 60 hrs]) the sequentially infected samples showed down regulation of lys-7 (Fig. 5a, b). These results suggest that, after S. aureus preinfection, the host had insufficient immune defenses to withstand a new invading pathogen. The F08G5.6 gene belongs to the family of CUB-like proteins and Daf-16 pathway regulated genes. qPCR analysis showed upregulation of this gene only in the S. aureus control; it was down regulated in both the sequentially infected samples and the P. mirabilis controls (Fig. 5a, c) indicating its specific role during pathogenesis. ATF-7 functions as a transcriptional regulator of PMK-1 MAPK–mediated innate immunity, acting as a repressor of immune gene expression but switching to an activator role upon activation by PMK-1. Sequentially infected samples showed downregulation of transcription factor ATF-7 (Fig. 5d) as compared with S. aureus and P. mirabilis controls (Fig. 5a). Recent reports have indicated that Daf-16 functions as a transcription factor of the insulin/insulin-like growth factor-1-mediated signaling pathway (40–42). Our data revealed upregulation of daf-16 in the controls (S. aureus[4, 8 and 12 hrs] and P. mirabilis[20 h and 60 h]) but significant down regulation in the sequentially infected samples (Figs. 5a, 6a). It is possible that the down regulation of these transcription factors results in failure of production of antimicrobial peptides that in turn leads to the host succumbing to the pathogen. Another anti-microbial peptide neuropeptide-like protein, nlp-29 showed upregulation in sequentially infected samples compared with controls (Figs. 5a, 6b). Interestingly, nlp-29 is the only candidate gene we identified as being upregulated after sequential infection by two pathogens and therefore needs further evaluation.

As one type of pathogen recognition receptor, C-type lectins are involved in innate immunity by recognizing and binding to carbohydrates exposed on the surfaces of microorganisms (43). In the present study, we analyzed the degree of expression of C-type lectin family genes, clec-60 and clec-87 during the different exposures times to S. aureus and P. mirabilis. clec-60 showed upregulation in both sequentially infected samples and their respective controls (Figs. 5a, 6c). Hence, it appears that clec-60 might actively participate in S. aureus and P. mirabilis recognition. However, clec-87 was down regulated during sequential infections (Fig. 6d), suggesting that it is not responsible for recognition of P. mirabilis during polymicrobial infections.

To explore the collective C. elegans immune response against sequential infections, we studied the regulation of a representative candidate antimicrobial genes from various regulatory pathways, namely daf-16 from the DAF-2/DAF-16 pathway (39), F08G5.6 from the DAF-2/DAF-16 and p38 MAPK pathways (30, 31), lys-7 from the DAF-2/DAF-16 and DBL-1/transforming growth factor-β pathways (26), nlp-29 and ATF-7 from the p38 MAPK pathway (26, 32) and clec-60 and clec-87, which are likely regulated by all three of the above immune pathways (26). Samples isolated from the controls displayed induction of all three immune pathway genes (Fig. 8). However, the sequentially infected samples showed down regulation of all the immune genes except nlp-29 (Figs. 6b, 8) and clec-60 (Figs. 6c, 8). These results collectively suggest the surrendering of that the innate immune system of C. elegans succumbs to sequential infection with the two pathogens studied.

image

Figure 8. Overall regulation of selective antimicrobial genes during sequential infection of C. elegans.

Download figure to PowerPoint

To study the expression pattern of non-antimicrobial peptides (i.e., unrelated peptide genes) during sequential infections, we studied the expression patterns of clk-2 and lbp-5. Our data indicate that the degree of expression of these unrelated genes does not change significantly during sequential. Both sequentially infected samples and their respective controls showed similar expression (Fig. 7).

The experiments presented here demonstrate that exposure of C. elegans to S. aureus and P. mirabilis induces its innate immune system to stave off these bacterial infections by regulating its antimicrobial genes, but that it cannot withstand sequential infection with both these pathogens. Our findings demonstrate that prior S. aureus infection of C. elegans makes this host vulnerable to succumbing to subsequent infection with P. mirabilis by compromising its immune system. Further studies are needed to confirm the role and contribution of other immune regulatory genes during the sequential infection with S. aureus and P. mirabilis.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

We thank the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health, National Center for Research Resources, for providing C. elegans N2 WT strain and E. coli OP50. Dr. K. Balamurugan thankfully acknowledges the Department of Biotechnology, Indian Council of Medical Research, Department of Science and Technology and Council of Scientific and Industrial Research (Senior Research Fellowship to GJ), New Delhi, India for financial assistance. The authors gratefully acknowledge the computational and bioinformatics facility provided by the Alagappa University Bioinformatics Infrastructure Facility (funded by DBT, GOI; Grant No. BT/BI/25/001/2006).

DISCLOSURE

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

The authors have no conflicting financial interests.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES
  • 1
    Bakaletz L.O. (2004) Developing animal models for polymicrobial diseases. Nat Rev Microbiol 2: 55268.
  • 2
    Kroes I., Lepp P.W., Relman D.A. (1999) Bacterial diversity within the human subgingival crevice. Proc Natl Acad Sci USA 96: 14,547–52.
  • 3
    Mylonakis E., Casadevall A., Ausubel F.M. (2007) Exploiting amoeboid and nonvertebrate animal model systems to study the virulence of human pathogenic fungi. PLoS Pathog 3: e101.
  • 4
    Darby C., Cosma C.L., Thomas J.H., Manoil C. (1999) Lethal paralysis of Caenorhabditis elegans by Pseudomonas aeruginosa. Proc Natl Acad Sci USA 96: 15,202–7.
  • 5
    Mahajan-Miklos S., Tan M.W., Rahme L.G., Ausubel F.M. (1999) Molecular mechanisms of bacterial virulence elucidated using a Pseudomonas aeruginosa-Caenorhabditis elegans pathogenesis model. Cell 96: 4756.
  • 6
    Tan M.W., Ausubel F.M. (2000) Caenorhabditis elegans: a modelgenetic host to study Pseudomonas aeruginosa pathogenesis. Curr Opin Microbiol 3 : 2934.
  • 7
    Aballay A., Ausubel F.M. (2001) Programmed cell death mediated byced-3 and ced-4 protects Caenorhabditis elegans from Salmonella typhimurium-mediated killing. Proc Natl Acad Sci USA 98 : 27359.
  • 8
    Aballay A., Yorgey P., Ausubel F.M. (2000) Salmonella Typhimurium proliferates and establishes a persistent infection in the intestine of Caenorhabditis elegans. Curr Biol 10 : 153942.
  • 9
    Labrousse A., Chauvet S., Couillault C., Kurz C.L., Ewbank J.J. (2000) Caenorhabditis elegans is a model host for Salmonella typhimurium. Curr Biol 10 : 154345.
  • 10
    Garsin D.A., Sifri C.D., Mylonakis E., Qin X., Singh K.V., Murray B.E., Calderwood S.B., Ausubel F.M. (2001) A simple model host for identifying Gram-positive virulence factors. Proc Natl Acad Sci USA 98 : 10,892–97.
  • 11
    Sifri C.D., Begun J., Ausubel F.M., Calderwood S.B. (2003) Caenorhabditis elegans as a model host for Staphylococcus aureus pathogenesis. Infect Immun 71 : 220817.
  • 12
    Peleg A.Y., Tampakakis E., Fuchs B.B., Eliopoulos G.M., Moellering R.C. Jr., Mylonakis E. (2008) Prokaryote–eukaryote interactions identified by using Caenorhabditis elegans. Proc Natl Acad Sci USA 105 : 14,585–90.
  • 13
    Hodgkin J., Kuwabara P.E., Corneliussen B. (2000) A novel bacterial pathogen, Microbacterium nematophilum, induces morphological change in the nematode C. elegans. Curr Biol 10 : 161518.
  • 14
    O’Rourke D., Baban D., Demidova M., Mott R., Hodgkin J. (2006) Genomic clusters, putative pathogen recognition molecules, and antimicrobial genes are induced by infection of C. elegans with M. nematophilum. Genome Res 16 : 100516.
  • 15
    Vigneshkumar B., Pandian S.K., Balamurugan K. (2012) Regulation of Caenorhabditis elegans and Pseudomonas aeruginosa machinery during interactions. Arch Microbiol. 194 : 22942.
  • 16
    JebaMercy G., Pandian S.K., Balamurugan K. (2011) Changes in Caenorhabditis elegans life-span and selective innate immune genes during Staphylococcus aureus infection. Folia Microbiol 56 : 37380.
  • 17
    Sivamaruthi B., Ganguli A., Kumar M., Bhaviya S., Pandian S.K., Balamurugan K. (2011) Caenorhabditis elegans as a model for studying Cronobacter sakazakii ATCC BAA-894 pathogenesis. J Basic Microbiol 51 : 54049.
  • 18
    Griffith D.P., Musher D.M., Itin C. (1976) Urease: The primary cause of infection-induced urinary stones. Invest Urol 13 : 34650.
  • 19
    Endimiani A., Luzzaro F., Brigante G., Perilli M., Lombardi G., Amicosante G., Rossolini G.M., Toniolo A. (2005) Proteus mirabilis bloodstream infections: risk factors and treatment outcome related to the expression of extended-spectrum beta-lactamases. Antimicrob Agents Chemother 49 : 259805.
  • 20
    Durai D., Pandian S.K., Balamurugan K. (2011) Establishment of a Caenorhabditis elegans infection model for Vibrio alginolyticus. J Basic Microbiol 51 : 24352.
  • 21
    Morse D.P., Bass B.L. (1999) Long RNA hairpins that contain inosine are present in Caenorhabditis elegans poly(A)+ RNA. Proc Natl Acad Sci USA 96 : 604853.
  • 22
    Keston A.S., Brandt R. (1965) The fluorometric analysis of ultramicro quantities of hydrogenperoxide. Anal Biochem 11 : 15.
  • 23
    Wolff S.P. (1994) Ferrous ion oxidation in presence of ferric ion indicator xylenol orange for measurement of hydroperoxides. Methods Enzymol 233 : 18289.
  • 24
    Shapira M., Hamlin B.J., Rong J., Chen K., Ronen M., Tan M.W. (2006) A conserved role for a GATA transcription factor in regulating epithelial innate immune responses. Proc Natl Acad Sci USA 103 : 14,086–91.
  • 25
    Chavez V., Mohri-Shiomi A., Garsin D.A. (2009) Ce-Duox1/BLI-3 generates reactive oxygen species as a protective innate immune mechanism in Caenorhabditis elegans. Infect Immun 77 : 4983–89.
  • 26
    Alper S., McBride S.J., Lackford B., Freedman J.H., Schwartz1 D.A. (2007) Specificity and complexity of the Caenorhabditis elegans innate immune response. Mol Cell Biol 27 : 554453.
  • 27
    Bork P., Beckmann G. (1993) The CUB domain A widespread module in developmentally regulated proteins. J Mol Biol 231: 53945.
  • 28
    Kerry S., TeKippe M., Gaddis N.C., Aballay A. (2006) GATA transcription factor required for immunity to bacterial and fungal pathogens. PLoS ONE 1 : e77.
  • 29
    Mallo G.V., Kurz C.L., Couillault C., Pujol N., Granjeaud S., Kohara Y. (2002) Inducible antibacterial defense system in C. elegans. Curr Biol 12 : 120914.
  • 30
    Li J., Ebata A., Dong Y., Rizki G., Iwata T., Lee S.S. (2008) Caenorhabditis elegans HCF-1 functions in longevity maintenance as a DAF-16 regulator. PLoS Biol e6 : 187085.
  • 31
    Bolz D.D., Tenor J.L., Aballay A. (2010) A conserved PMK-1/p38 MAPK is required in C. elegans tissue specific immune response to Y. pestis infection. J Biol Chem 285 : 10,832–40.
  • 32
    Shivers R.P., Pagano D.J., Kooistra T., Richardson C.E., Reddy K.C. (2010) Phosphorylation of the conserved transcription factor ATF-7 by PMK-1 p38 MAPK regulates innate immunity in Caenorhabditis elegans. PLoS Genet 6 : e1,000,892.
  • 33
    Nathoo A.N., Moeller R.A., Westlund B.A., Hart A.C. (2001) Identification of neuropeptide-like protein gene families in Caenorhabditis elegans and other species. Proc Natl Acad Sci USA 98 : 14,000–5.
  • 34
    Pujol N., Cypowyj S., Ziegler K., Millet A., Astrain A., Goncharov A., Jin Y., Chisholm A.D., Ewbank J.J. (2008) Distinct innate immune responses to infection and wounding in the C. elegans epidermis. Curr Biol 18 : 4819.
  • 35
    Franc N.C., White K. (2000) Innate recognition systems in insect immunity and development: New approaches in Drosophila. Microbes Infect 2 : 24350.
  • 36
    Gartner A., Milstein S., Ahmed S., Hodgkin J., Hengartner, M.O. (2000) A conserved checkpoint pathway mediates DNA damage-induced apoptosis and cell cycle arrest in C. elegans. Mol Cell 5 : 43543.
  • 37
    Plenefisch J., Xiao H., Mei B., Geng J., Komuniecki P.R., Komuniecki R.W. (2000) Secretion of a novel class of iFABPs in nematodes: coordinate use of the Ascaris/Caenorhabditis model systems. Mol Biochem Parasitol 105 : 22336.
  • 38
    Bogaerts A., Beets I., Schoofs L., Verleyen P. (2010) Antimicrobial peptides in Caenorhabditis elegans. Invertebrate Survival Journal 7 : 4552.
  • 39
    Rahman M.M., Stuchlick O., El-Karim E.G., Stuart R., Kipreos E.T., Wells L. (2010) Intracellular protein glycosylation modulates insulin mediated lifespan in C. elegans. Aging 2 : 67890.
  • 40
    Ogg S., Paradis S., Gottlieb S., Patterson G.I., Lee L., Tissenbaum H.A., Ruvkun G.B. (1997) The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 389 : 99499.
  • 41
    Garigan D., Hsu A.L., Fraser A.G., Kamath R.S., Ahringer J., Kenyon C. (2002) Genetic analysis of tissue aging in Caenorhabditis elegans: a role for heat-shock factor and bacterial proliferation. Genetics 161: 110112.
  • 42
    Kwon E.S., Narasimhan S.D., Yen K., Tissenbaum H.A. (2010) A new DAF-16 isoform regulates longevity. Nature 466 : 49802.
  • 43
    Drickamer K. (1993) Evolution of Ca (2+)-dependent animal lectins. Prog Nucleic Acid Res Mol Biol 45 : 20732.