Effect of srtA and srtB gene expression on the virulence of Staphylococcus aureus in animal models of infection

William J. Weiss1,*, Eileen Lenoy1, Timothy Murphy1, LuAnna Tardio1, Pamela Burgio1, Steven J. Projan1, Olaf Schneewind2 and Lefa Alksne1

1 Wyeth Research, 401 N. Middletown Rd., Pearl River, NY 10965; 2 Committee on Microbiology, University of Chicago, Chicago, IL, USA

Received 20 August 2003; returned 29 October 2003; revised 11 November 2003; accepted 17 November 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective: The role that the surface proteins anchored by the srtA and srtB gene products play in the ability of Staphylococcus aureus bacteria to establish infection was investigated in several animal models.

Methods: Wild-type and corresponding mutants with deletions of the srtA and/or srtB genes were used in murine acute lethal infection, septic arthritis, kidney infection and rat endocarditis models.

Results: The LD50 of the wild-type and srtB– knockout were comparable and approximately two- to four-fold lower than the required inoculum of the srtA– and srtA–B strains. This difference was exhibited as a two-fold greater mortality at the highest inoculum. The wild-type strain established arthritic inflammation in over 90% of the animals with a maximum arthritic index of 6.5 by days 17–21. The srtB knockout was able to cause inflammation in 70–80% of the mice, but with a lower index of 3.0. Both the srtA– and srtA–B– strains appeared to be less virulent in this model with arthritic indices of around 0.5 and only 20% of the animals with inflammation. Strains with the srtA mutation achieved statistically significant lower titres than wild-type in kidneys of mice after intravenous infection. Mean bacterial counts in cardiac vegetations were significantly higher for the wild-type and srtB– strain compared with the srtA and srtA–B– strains.

Conclusion: Results from this study substantiate the role of the srtA gene product in the establishment of infections and further studies are warranted to define and exploit this as a target for antimicrobial chemotherapy.

Keywords: sortase, virulence, arthritis, endocarditis


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The surface of Gram-positive bacteria contains proteins that interact with host tissues and facilitate expression of virulence.1 Many of these surface proteins are anchored to the cell wall via a cell C-terminal sorting signal reaction.2 During this reaction in Staphylococcus aureus, the sortase enzyme encoded by srtA is reported to cleave between the threonine and glycine of the LPXTG motif and allow the covalent anchoring of the protein to the bacterial cell wall via a transpeptidation reaction.35 Proteins such as protein A, clumping factor and fibronectin-binding proteins are attached to the cell wall by this type of reaction.6 Expression of these cell-wall-associated proteins enables binding to host tissues resulting in colonization and infection.7 Proteins anchored by this mechanism also serve to block phagocytosis, act as adhesins, agglutinins and promote interaction with host-cell membranes.

S. aureus, a Gram-positive organism, is a human pathogen of increasing importance. It is responsible for numerous infections and syndromes ranging in severity from skin and soft tissue infections to endocarditis and septic arthritis.8 S. aureus is prevalent in the hospital setting and represents an important cause of health-care associated infections. Increased drug resistance among S. aureus has been observed and now includes the first fully vancomycin-resistant S. aureus isolate.9 The prevalence of these isolates, their virulence and increasing resistance indicates the need for alternative therapy to treat these infections.10

Earlier studies of sortase-mediated virulence have only examined the srtA gene, however it has been predicted that more than one sortase orthologue is present in the genomes of Gram-positive bacteria.11,12 Recent studies in both S. aureus13 and Streptococcus pyogenes have suggested that the proteins encoded by srtA and srtB have different substrates for transpeptidation. Mazmanian et al.13 reported that srtB in S. aureus is part of an iron-regulated operon and recognizes an NPQTN motif in one cell-surface-expressed protein. Researchers examining the role of an srtB homologue in S. pyogenes have found that its protein product appears to differentially anchor an LPXTG-containing protein, T6.14 Furthermore, they have found that the gene srtB is not universally present among the genus Streptococcus. These results indicate that among the sortase homologues it is srtA that has the most significant effect on early events in colonization. Although the effect of srtA inactivation on the virulence and host colonization of a number of Gram-positive bacteria has been analysed in a number of other animal models,1520 this study is the first to directly compare the effects of mutations in both srtA and srtB in a number of animal models of infection.

To explore the role and importance of the sortase enzyme in anchoring these cell wall proteins and their role in the virulence of S. aureus, we have compared wild-type and various isogenic sortase- deficient mutants in several animal infection models which focus attention on different aspects of bacterial–host interaction. The acute lethal infection model is used to represent a systemic bacteraemia of overwhelming infection. The rat model of endocarditis relies on the ability of the organism to adhere to an implanted cannula surrounded by a fibrin and platelet clot, establishing an infection and continuing to grow within this matrix to form a deep seeded bacterial vegetation. Although progression of bacterial infection from the bloodstream to joints to cause septic arthritis is unknown, several adhesion molecules are of importance among them the collagen-binding adhesion molecules.21,22 The ability of these strains, either wild-type or mutants in sortase expression, to establish and maintain an infection in these animal models will validate sortase as a potential target for antibacterial therapeutic agents.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Strains

S. aureus strain Newman,23 a human clinical isolate, represents the wild-type strain in these studies. The isogenic sortase knockout mutants: SKM12—sortase A deletion (srtA–), SKM7—sortase B deletion (srtB–) and SKM14—sortase A and B deletions (srtA–B–) were generated by allelic exchange as previously described.13 All strains were obtained from Dr Olaf Schneewind, University of Chicago, Chicago, IL, USA.

S. aureus clumping assay

Stationary cultures were diluted and grown in brain heart infusion broth to exponential phase by incubating in a shaking incubator at 37°C for 5.5 h. Samples were then removed from the incubator and centrifuged at 14 000 rpm for 3 min at 4°C. Excess supernatant was aspirated off taking care not to disturb the pellet. Pellets were resuspended in a fibrinogen solution consisting of 1 mg/mL fibrinogen (Sigma Chemical Co., St Louis, MO, USA; Fraction I, Type I-S, from bovine plasma) in phosphate buffered salt solution (Cellgro brand, Dulbecco’s PBS 1x, no calcium, no magnesium). Absorbance measurements were taken for each sample at 600 nm immediately and at 45 and 90 min after resuspension in the fibrinogen solution. The percent change in absorbance is determined by dividing the absorbance at the time points by that obtained at the initial time point multiplied by 100. In the presence of an active sortase protease, clumping factor protein anchored in the cell wall by sortase actively recognizes and binds to fibrinogen in solution, forming aggregation or ‘clumps’ which fall out of solution and are traced by a decreasing OD over time. These clumps can be visually observed by mixing cells scraped from the solid medium into fibrinogen on a cover slide.

Acute lethal infection model

Female mice, strain CD-1 (Charles River Laboratories, Kingston, NY, USA) 20 ± 2 g each, were challenged by intraperitoneal injection of bacteria from 5 h broth cultures suspended in 5% hog gastric mucin.24 Five animals were infected at each of three inoculum levels for each organism of 105, 106 and 107 colony forming units (cfu) per animal. Bacterial concentrations administered were determined by plate counts carried out on the inoculum. The 7 day survival ratios from three separate tests were pooled for estimation of the median lethal dose (LD50) by a computerized program for probit analysis.25

Septic arthritis model

Fifteen female NMRI mice, 5–8 weeks old (Charles River Laboratories), were infected intravenously with 0.2 mL of bacterial suspension (106–107 cfu) contained in phosphate buffered saline (PBS) for each of the four strains tested.26 The inoculum was prepared by picking colonies from trypticase soy agar plates incubated for 24 h. Plate counts were carried out before and with the actual inocula to both verify and equilibrate the inocula with each organism. The mice were then observed daily for the duration of the experiment (up to 21 days). Daily observations included recording swelling/erythema of the talocrural (ankle) and radiocarpal (wrist) joints. The appearance was scored for each joint (right and left ankle and wrist) according to the following scale: 0 = normal appearance, 1 = mild swelling, 2 = mild to moderate swelling, 3 = moderate to marked swelling and 4 = severe swelling with erythema. The observed scores for all four limbs were added together for all animals and divided by the number of surviving animals in that group to determine the arthritic index for each day.

Kidney infection model

Six- to eight-week-old female C3H/HeJ mice (Charles River Laboratories) (n = 10 per group) were infected by direct bladder injection of 107 cfu of S. aureus Newman, SKM7, SKM12 or SKM14 diluted in PBS taken from TSA plates. Inoculum infection concentrations were verified by plate counts. Four days after infection, mice were euthanized and kidneys excised, homogenized, diluted in normal saline and plated in duplicate for determination of cfu.

Rat endocarditis model

The rat model of experimental endocarditis has been previously described.27 Briefly, endocarditis was produced in male Sprague–Dawley rats (250 ± 25 g; Charles River Laboratories) by insertion of a sealed polyethylene catheter (PE10) through the right carotid artery into the left ventricle and sutured in place as a point of adherence for bacterial infection. At 48 h after implantation of the catheter, a 5 h bacterial culture of Newman (wild-type; wt), srtB– (SKM7), srtA– (SKM12) or srtA–B (SKM14) was diluted to 105 cfu/mL in sterile saline and 1 mL was injected intravenously into the tail vein of 10 rats per organism. Inoculum infection concentrations were verified by plate counts. Hearts were aseptically removed 2 days after infection, homogenized and serially diluted in saline for determination of bacterial titres, expressed as log10 cfu per heart.

Animal disposition

All procedures involving the use of laboratory animals were carried out in accordance with AAALAC standards, current standards stated in the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996) and were approved by an Animal Care and Use Committee review.

Statistical analysis

For the acute lethal infection model, the 7 day survival ratios from three separate tests were pooled for estimation of the median lethal dose (LD50) by a computerized program for probit analysis (SAS version 8.1). Endocarditis and kidney infection model tests were analysed using mixed-model analysis of variance (ANOVA) methods or the Kruskal–Wallis test. The counts of cfu in cardiac vegetations or kidneys were used for a comparison of statistical significance compared to the wild-type control groups using a two-tailed t-test. The results for the arthritis model, both arthritic index and percentage of animals with inflammation, were compared to each other over the course of the experiment using the GENMOD procedure in SAS version 8.1 fitting a model for repeated ordered categorical response data.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
S. aureus Newman (wt), SKM7 (srtB–), SKM12 (srtA–) and SKM14 (srtA–B–) were evaluated in the fibrinogen-mediated clumping assay (Figure 1). In the presence of an active sortase transpeptidase, clumping factor proteins anchored on the cell surface by sortase recognize and bind to fibrinogen in solution, forming a clearly visible aggregation which falls out of solution and is traced by a decreasing OD600 over time. Bacterial cells lacking a functional srtA gene do not aggregate with fibrinogen because the absent sortase cannot facilitate the anchoring of clumping factor on the cell surface. Cells therefore remain in suspension over time, as measured by a steady OD600. The results of the clumping assay are presented graphically in Figure 2. The data indicate a 40% reduction in OD from the initial reading by 45 min for the wild-type and srtB– isolates. This increased to a 70% reduction by 90 min, indicating significant clumping and precipitation of the bacteria–fibrinogen complex resulting in this change in absorbance. In contrast, the srtA– and srtAB– strains demonstrated minimal change in OD at 45 min and only a 37% and 20% reduction at 90 min.



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Figure 1. Clumping factor proteins anchored in the cell wall by sortase actively recognize and bind to fibrinogen in solution, forming aggregation or ‘clumps’ (indicated by arrows). Pictured here are Staphylococcus aureus: (a) Newman wild-type strain, (b) SKM12 srtA– knockout; (c) SKM7 srtB– knockout and (d) SKM14 srtA–B– knockout.

 


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Figure 2. Effect of srtA and srtB gene expression on the ability of strains to clump with fibrinogen and cause change in OD over time (mean value ± S.D.).

 
Each of the strains tested, wild-type and sortase knockouts, were capable of inducing mortality in mice following intraperitoneal injection (Figure 3). Inoculum levels of 5–8 x 105 cfu did not cause death in any of the mice infected with the strains. Increasing the infection inoculum to 107 cfu resulted in greater mortality for the wild-type and srtB– strains (73% and 80%, respectively) than the srtA– and the srtA–B strains (36% and 43%, respectively). A probit analysis was carried out to compare the LD50s of the different strains in the study. Fiducial confidence intervals were constructed using the procedure Probit in SAS version 8.1. The LD50s and associated 95% confidence intervals are reported in Table 1. The LD50 of the wild-type (Newman) strain was not statistically significantly (P > 0.05) different from the LD50s of the srtB– and srtA–B strains, but was statistically significantly (P < 0.05) lower than that of the srtA– strain. The lack of a statistically significant difference between the wild-type (Newman) and the srtAB– (SKM14) strains was the result of a slight overlap between the upper bound and lower bound confidence limits of these two strains, respectively. The LD50 of the srtB strain was statistically significantly (P < 0.05) lower than the LD50s of the srtA– and srtA–B– strains. Absence of the srtA-encoded enzyme requires a greater number of bacteria in the infection inoculum to elicit the same response, as measured by mortality, as either the wild-type or srtB– knockout strains.



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Figure 3. Effect of sortase expression on survival of mice from an acute lethal infection with wild-type and sortase knockout mutants of Staphylococcus aureus. Seven day survival determined from pooled data from three separate tests after intraperitoneal injection of bacterial inoculum (n = 5/inoculum level).

 

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Table 1. Median lethal dose (LD50) of wild-type and sortase knockout mutant S. aureus (n = 5/inoculum level) in murine acute lethal infection model
 
S. aureus Newman caused arthritis exhibited as joint inflammation in 60%, 80% and 100% of the animals on days 5, 10 and 17, respectively (Figure 4). The srtB– knockout strain SKM7 demonstrated comparable results with arthritis in 46%, 65% and 60% of the infected animals on the same days, respectively. In contrast, SKM12 and SKM14, the srtA– and srtAB– knockouts induced inflammation in only approximately 20% of the animals infected throughout the course of the study. The arthritic index was used as a measure of the severity and extent of the infection in individual animals (Figure 5). Mice infected with wild-type S. aureus exhibited an increase in the arthritic index over time with maximum indices of 6.0–6.5 on days 17–21. SKM7 (srtB–) which induced arthritis in a slightly lower percentage of animals exhibited lower indices with a maximum index of 3.0 on day 17. The srtA– and srtAB knockouts both showed arthritic indices of only 0.5–1.0 for days 5–21. Throughout the course of the 21 day study, mortality was observed for 26% (four of 15) of the Newman infected mice and 33% (five of 15) of the SKM7 (srtB–) infected mice whereas all the SKM12 (srtA–) and SKM14 (srtA–B–) mice survived.



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Figure 4. Ability of wild-type Staphylococcus aureus Newman or sortase-deficient strains to induce arthritis in NMRI mice (n = 15).

 


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Figure 5. Severity of arthritis in NMRI mice inoculated with 106–107 cfu of Staphylococcus aureus Newman or sortase-deficient mutants (n = 15). Data represented as means ± S.D.

 
The results of the kidney infection model for the wild-type and mutant S. aureus demonstrated further differences between the strains (Figure 6). Mean kidney titres ± S.D. were: Newman, 5.5 ± 2.2; SKM7 (srtB–), 4.2 ± 1.6; SKM12 (srtA–), 3.5 ± 2.1; and SKM14 (srtAB–), 3.1 ± 1.1 log10 cfu/kidney pair, respectively. The differences between the SKM12 and SKM14 strains, both with the srtA mutation, and the Newman strain were considered to be statistically significant (P < 0.06 and P < 0.01, respectively). SKM7, with a mutation in srtB, was not considered different from the Newman strain.



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Figure 6. Recovery of Staphylococcus aureus strains from kidneys of C3H/HeJ mice 4 days after intravenous injection of wild-type and sortase-deficient mutants (n = 10). Each individual animal is indicated by a circle symbol with mean bacterial titres represented as a line.

 
Infection in cardiac vegetations was achieved by all strains 48 h after intravenous infection of a comparable inoculum to instrumented rats (Figure 7). Counts for the Newman strain ranged from 5 to 9.2 log10 cfu/heart with a mean bacterial titre of 7.7 ± 1.6 log10 cfu. SKM7, the srtB– knockout exhibited comparable titres to the wild-type strain with a range of 4.2–8.7 log10 cfu and a mean titre of 7.3 ± 1.7 log10 cfu. Both the SKM12 and SKM14 strains achieved lower cardiac titres with mean counts of 5.4 ± 1.9 and 4.4 ± 2.2 log10 cfu, respectively. These were considered to be statistically significant from both the Newman (P <= 0.01 and 0.002) and the SKM7 (P <= 0.03 and 0.005) strains.



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Figure 7. Bacterial titres in cardiac vegetations of rats at 48 h after intravenous inoculation of S. aureus Newman and sortase-deficient mutants to catheter implanted Sprague–Dawley rats (n = 10). Each individual animal is indicated by a circle symbol with mean bacterial titres represented as a line.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It has been more than a decade since the first recognition of a conserved anchoring motif containing the sequence ‘LPXTG’ among cell surface proteins in Gram-positive bacteria.28 Since that time the function of these proteins in the virulence of a number of important pathogens has been examined.29,30 Many of these proteins (termed MSCRAMMS for microbial surface components recognizing adhesive matrix molecules) promote the adherence of both pathogenic and non-pathogenic bacteria to host matrix molecules, enabling the establishment of a niche for the organism. Others, such as protein A, allow the bacteria to evade innate host defences. Subsets of LPXTG- containing proteins have been shown to be important in bacterial association with different host tissues. For example, clumping factor has been shown to be important in the establishment of endocarditis,31 and collagen adhesin is involved in the establishment of arthritis,32 whereas internalin A is an LPXTG-motif-containing protein required for the internalization of Listeria monocytogenes. In every case where it has been studied, the cell-wall presentation of these factors is mediated by the transpeptidase sortase33 suggesting that this enzyme plays a critical role in the establishment and perhaps maintenance of bacterial infection.

These results of the in vitro clumping assay demonstrate that SrtA (protein) is responsible for the anchoring of the clumping factor proteins to the cell surface. In the absence of srtA, the strain is impaired in its ability to bind fibrinogen in solution. The absence of the srtB gene alone does not appear to affect the ability of the strain to bind fibrinogen.

The results of our acute lethal infection model show less than a log difference in the LD50 between all the strains. However, when considering survival, we observe two-fold greater mortality with the wild-type and srtB– isolates compared to the srtA– and srtA–B–isolates. The nature of this model relies on the transport of bacteria from the peritoneum to the bloodstream via the lymphatics. Bacteria exhibit growth curves within the animal similar to those observed in vitro with an exponential phase followed by a stationary phase.34 The growth of the organism, both in tissues and the blood, induces a host immune response. The ability to survive and cause a lethal infection therefore requires both tissue penetration and evasion of the host defences. The similarity of the median lethal dose (LD50) required for each of the strains is an indication that lethality can be achieved when the infection reaches an overwhelming bacterial titre. Differences seen for mortality at comparable inoculum can be attributed to the ability to invade host tissue and evade the host’s defence mechanisms.

Non-bacterial thrombotic endocarditis was induced by the insertion of a polyethylene catheter into the heart of experimental animals. This is prompted by the deposition of fibrin and platelets at the catheter site producing macroscopic aseptic lesions that are susceptible to colonization by bacteria in the bloodstream. We observed statistically significant differences between mean cardiac bacterial counts for the wild-type Newman strain and strains with the srtA– knockout (srtA– and srtA–B–) in this model. It appears that the srtA gene deletion and ensuing loss of the ability to anchor certain surface proteins, such as those responsible for fibrin binding, has affected the ability of the srtA– strains to infect and colonize in the cardiac vegetation. The strain with only the srtB deletion was not affected in its ability to establish cardiac infection. Within the context of this experimental model, the presence of active sortase encoded by srtA enables the bacteria to achieve a greater titre within the vegetation at the time of sampling. Additional testing should be carried out to determine the effect of active sortase enzyme on bacterial titres over time and the effect on mortality from endocarditis.

Collagen adhesion, as well as fibrinogen-binding proteins, are believed to play an important role as virulence factors in infective arthritis.35 They appear to mediate adherence of the circulating bacteria to the target tissue of the joints. We have observed a significant effect on the ability of srtA and srtA–B– knockouts to establish arthritis and on the severity of the infection in the few animals that appeared infected. The virulence factors for establishing infection appear to be anchored by the sortase enzyme encoded by the srtA gene. The srtB–knockout strain established arthritis of moderate severity in over 60% of the mice infected. Although this was less than for the wild-type strain, it was significantly more and of greater severity than those strains with the srtA deletion. These results support those previously observed with the wild-type Newman strain and an isogenic sortase- deficient variant strain, SKM3.18 They also observed decreased frequency of arthritis as well as a lower arthritic index for mice inoculated with the sortase-mutant strain.

Recent studies13,36 have proposed that srtB is required for staphylococcal persistence during infection. Results of these studies indicate that although mutants of srtB establish infection in renal abscesses and arthritic joints that are equivalent to the wild-type strain, there was a significant reduction in infective titre over time compared to controls. We have observed in our study that the srtB deficient strain was able to establish systemic, arthritic, cardiac and renal infections comparable to wild-type. Additional testing would be required in these models to determine the role of srtB in the persistence of these infections.

When considering all the data, the sortase enzyme encoded by the srtA gene, appears to play a major role in the establishment of staphylococcal bacterial infections both systemic and localized. Further studies are therefore warranted to identify agents that are active at inhibiting sortase and would serve as an alternative for antimicrobial chemotherapy.


    Acknowledgements
 
We gratefully acknowledge the technical assistance of Christine Schuller, statistical analysis by Jorge Quiroz and manuscript review by C. Hal Jones.


    Footnotes
 
* Corresponding author. Tel: +1-845-602-2719; Fax: +1-845-602-5671; E-mail: weissw{at}wyeth.com Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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