Contribution of Panton-Valentine Leukocidin in Community-Associated Methicillin-Resistant Staphylococcus aureus Pathogenesis

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains typically carry genes encoding Panton-Valentine leukocidin (PVL). We used wild-type parental and isogenic PVL-deletion (Δpvl) strains of USA300 (LAC and SF8300) and USA400 (MW2) to test whether PVL alters global gene regulatory networks and contributes to pathogenesis of bacteremia, a hallmark feature of invasive staphylococcal disease. Microarray and proteomic analyses revealed that PVL does not alter gene or protein expression, thereby demonstrating that any contribution of PVL to CA-MRSA pathogenesis is not mediated through interference of global gene regulatory networks. Inasmuch as a direct role for PVL in CA-MRSA pathogenesis remains to be determined, we developed a rabbit bacteremia model of CA-MRSA infection to evaluate the effects of PVL. Following experimental infection of rabbits, an animal species whose granulocytes are more sensitive to the effects of PVL compared with the mouse, we found a contribution of PVL to pathogenesis over the time course of bacteremia. At 24 and 48 hours post infection, PVL appears to play a modest, but measurable role in pathogenesis during the early stages of bacteremic seeding of the kidney, the target organ from which bacteria were not cleared. However, the early survival advantage of this USA300 strain conferred by PVL was lost by 72 hours post infection. These data are consistent with the clinical presentation of rapid-onset, fulminant infection that has been associated with PVL-positive CA-MRSA strains. Taken together, our data indicate a modest and transient positive effect of PVL in the acute phase of bacteremia, thereby providing evidence that PVL contributes to CA-MRSA pathogenesis

Rapid Neutrophil Destruction following Phagocytosis of Staphylococcus aureus

Mechanisms underlying the enhanced virulence phenotype of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are incompletely defined, but presumably include evasion of killing by human polymorphonuclear leukocytes (PMNs or neutrophils). To better understand this phenomenon, we investigated the basis of rapid PMN lysis after phagocytosis of USA300, a prominent CA-MRSA strain. Survival of USA300 clinical isolates after phagocytosis ultimately resulted in neutrophil lysis. PMNs containing ingested USA300 underwent morphological changes consistent with apoptosis, but lysed rapidly thereafter (within 6 h), whereas cells undergoing FAS-mediated apoptosis or phagocytosis-induced cell death remained intact. Phagosome membranes remained intact until the point of PMN destruction, suggesting lysis was not caused by escape of S. aureus from phagosomes or the cytolytic action of pore-forming toxins. Microarray analysis of the PMN transcriptome after phagocytosis of representative community-associated S. aureus and healthcare-associated MRSA strains revealed changes unique to community-associated S. aureus strains, such as upregulation of transcripts involved in regulation of calcium homeostasis. Collectively, the data suggest that neutrophil destruction after phagocytosis of USA300 is in part a form of programmed necrosis rather than direct lysis by S. aureus pore-forming toxins. We propose that the ability of CA-MRSA strains to induce programmed necrosis of neutrophils is a component of enhanced virulence

Co-infection experiments with USA300 or USA400 parental or isogenic Δ<i>pvl</i> mutant strains assayed at end stages of bacteremia.

1<p>Competition assays were used to compare three wild type-Δ<i>pvl</i> mutant pairs: LAC-LACΔ<i>pvl</i> (n = 28), SF8300-SF8300Δ<i>pvl</i> (n = 17), and MW2-MW2Δ<i>pvl</i> (n = 25), where n is the total number of animals used in each experiment. A 1∶1 mixture containing approximately 3×10⁷ CFUs of wild type parent and 3×10⁷ CFUs of isogenic Δ<i>pvl</i> mutant were used to co-infect New Zealand white rabbits via the marginal ear vein. Mean bacterial densities comprising of both wild type and Δ<i>pvl</i> mutant from vital organs and blood are shown. The competition index (CI), which is the logarithm (log₁₀) of the output ratios of parent and isogenic mutant after correction for variations in input ratios, are shown. A positive CI value indicates enhanced tissue infectivity of the parent, whereas a negative CI value indicates enhanced tissue infectivity of the mutant; a CI = 0 is the no-effect value.</p>2<p>The null hypothesis (CI = 0) that there was no difference in bacterial densities between parent and isogenic Δ<i>pvl</i> mutant in rabbit vital organs was tested using a paired Student's <i>t</i> test. All two-tailed <i>P</i> values were not statistically significant (<i>P</i>>0.05).</p

TaqMan real-time RT-PCR analysis reveals that PVL does not alter <i>agr</i>-regulated transcripts in USA300 and USA400 clinical strains.^±

±<p>TaqMan real-time RT-PCR was performed as described in Methods. Results are expressed as the mean fold-change of 3–5 experiments (exponential growth, Exp.) or 4–7 experiments (stationary phase of growth, Stat.) with one exception<i>^a</i> (one of the TaqMan reactions failed, n = 2). The relative expression level of each transcript (dCT) was compared in parent vs. Δ<i>pvl</i> strains using a paired Student's <i>t</i>-test (^*<i>P</i><0.05 versus Δ<i>pvl</i>). Except for <i>lukF-PV</i>, none of the transcripts in any of the strains met standard criteria required for differentially expressed genes (>2-fold change in the wild-type vs. Δ<i>pvl</i> mutant strain and <i>P</i><0.05).</p><p><i>agrA</i>, accessory global regulator; <i>hla</i>, alpha-toxin; <i>hlgA</i>, gamma-haemolysin component A; <i>splA</i>, serine protease; <i>spa</i>, protein A; <i>sdrD</i>, serine aspartate repeat protein; <i>clfB</i>, clumping factor B; and <i>lukF-PV</i>, Panton-Valentine leukocidin component F.</p

Time-course single-strain infection experiments with a USA300 parental or isogenic Δ<i>pvl</i> mutant strains.¹

1<p>Rabbits were euthanized and log₁₀CFU per gram of lung, spleen, and kidney were determined at 24, 48 and 72 hours post infection. It was not possible to conduct an experimental group at 96 hours post infection because rabbits loss >15% of the baseline weight by 72 hrs post infection, which is a moribund condition stipulated by UCSF animal use committee for euthanization. Two-sided <i>P</i> values by unpaired Student's <i>t</i> test are reported.</p