Identification of an intracellular M17 family leucine aminopeptidase that is required for virulence in Staphylococcus aureus

Abstract

Staphylococcus aureus is a highly virulent bacterial pathogen capable of causing a variety of ailments throughout the human body. It is a major public health concern due to the continued emergence of highly pathogenic methicillin resistant strains (MRSA) both within hospitals and in the community. Virulence in S. aureus is mediated by an array of secreted and cell wall associated virulence factors, including toxins, hemolysins and proteases. In this work we identify a leucine aminopeptidase (LAP, pepZ) that strongly impacts the pathogenic abilities of S. aureus. Disruption of the pepZ gene in either Newman or USA300 resulted in a dramatic attenuation of virulence in both localized and systemic models of infection. LAP is required for survival inside human macrophages and gene expression analysis shows that pepZ expression is highest in the intracellular environment. We examine the cellular location of LAP and demonstrate that it is localized to the bacterial cytosol. These results identify for the first time an intracellular leucine aminopeptidase that influences disease causation in a Gram-positive bacterium.

Figure 1. LAP is required for virulence in mice and survival within human macrophages

S. aureus wild type strain Newman and a Newman pepZ⁻ mutant were used to inoculate 10 mice each. A. Infected mice were monitored for weight loss over a twelve-day period. Percentage weight change for wild-type (□) and mutant (○) infected mice is plotted with statistical significance indicated by * and ** for p values >0.05 and >0.01 respectively. B. At 5 and 12 days post inoculation wild-type (shaded) and mutant (unshaded) infected mice limbs were observed and scored for clinical symptoms of arthritis resulting in an arthritic index score. Black lines indicate average scores, and p values for statistical significance are shown. C. Histological examination of mice 12 days post-inoculation for wild-type (●) and mutant (○) infected mice. Black lines indicate average index scores, and p values for statistical significance are shown. D. Bacterial dissemination to the kidneys for wild-type (shaded) and mutant (unshaded) infected was determined. Black lines indicate average loads, and p values for statistical significance are shown. E. A human infection model of clearance and survival was performed using primary cultured human macrophages and the Newman wild-type (black) and pepZ mutant (grey) strains. Error bars represent standard deviation. Significance (*) was determined by t test (p < 0.05).

Figure 2. Transcription profiling of the pepZ gene

A. The pepZ locus (to scale). B. Identification of the pepZ transcription start site by 5’ RACE. Shown in bold and underscore are the translation start site (ATG), the putative ribosome binding site (GGAGG), the +1 site (G), and putative −10 (TAcAAT) and −35 (gTGtCA) sequences for σ^A recognition. C and D. Analysis of pepZ-lacZ transcription fusion in Newman (C) and USA300 FPR (D). Wild type strains containing the pepZ-lacZ fusion were grown in TSB for 8 hours and samples taken for β–galactisodase assays at each hour (■). Following 3 h growth, bacteria were sub-cultured into fresh TSB (□) and 10% milk medium (○), with samples taken hourly for a further 5 hours. E. Analysis of pepZ-lacZ expression inside macrophages. Cultured RAW264.7 cells were infected with Newman (grey) and USA300 FPR pepZ-lacZ (black) fusion strains, with samples taken at the time-points indicated for β–galactisodase assays. For comparison the maximum levels of pepZ-lacZ expression in each strain in TSB is also shown. β–galactisodase assays were performed in triplicate. Error bars represent standard deviation. Statistical significance was determined by t-test (* = p < 0.05).

Figure 3. Competitive growth index for wild type strains versus pepZ⁻ mutants

Flasks of TSB were inoculated with equal amounts of wild type and pepZ⁻ mutant and allowed to grow for 7 days. Each day the number of wild type and pepZ⁻ mutant bacteria were determined and the competitive index calculated. Error bars represent the standard error between three replicate experiments. Significance was determined by t-test (*= p<0.05)

Figure 4. Western blot analysis of LAP to determine subceullular localization

Strain USA300 FPR containing a plasmid encoded, his-tagged LAP (lanes 1, 3 and 5) or an empty vector control (lanes 2, 4 and 6) were grown in TSB for the times specified. Samples were taken for western immunoblot analysis using an anti-his antibody.

Figure 5. LAP is required for the full virulence of CA-MRSA

A. Abscess model of infection. The USA300 FPR wild-type and pepZ⁻ mutant were used to subcutaneously inoculate 10 mice each, which were monitored for 7 days, with abscesses harvested and bacterial loads determined. The average cfu/abscess is indicated by a bold marker, significance (*) was determined using a Student’s t-test (p=0.029). B. Sepsis model of infection. USA300 FPR wild-type (■) and pepZ⁻ mutant (○) bacteria were used to intravenously inoculate 10 mice each via tail vein injection. Data is represented as percent survival over time, significance (*) was determined via a log rank test (p =1.3×10⁻⁵).