Lack of a major role of Staphylococcus aureus Panton-Valentine leukocidin in lower respiratory tract infection in nonhuman primates

Abstract

Panton-Valentine leukocidin (PVL) is a two-component cytolytic toxin epidemiologically linked to community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections, including serious invasive infections caused by the epidemic clone referred to as strain USA300. Although PVL has long been known to be a S. aureus virulence molecule in vitro, the relative contribution of this leukotoxin to invasive CA-MRSA infections such as pneumonia remains controversial. We developed a nonhuman primate model of CA-MRSA pneumonia and used it to test the hypothesis that PVL contributes to lower respiratory tract infections caused by S. aureus strain USA300. The lower respiratory tract disease observed in this monkey model mimicked the clinical and pathological features of early mild to moderate S. aureus pneumonia in humans, including fine-structure histopathology. In this experiment using a large sample of monkeys and multiple time points of examination, no involvement of PVL in virulence could be detected. Compared with the wild-type parental USA300 strain, the isogenic PVL deletion-mutant strain caused equivalent lower respiratory tract pathology. We conclude that PVL does not contribute to lower respiratory tract infection in this nonhuman primate model of human CA-MRSA pneumonia.

Figure 1

Gross and microscopic examination of cynomolgus macaque lungs infected with wild-type (left) or Δpvl (right) strains of USA300. Similar histopathology features were observed in specimens collected at 2-day (A–D), 4-day (E–H), and 8-day (I–L) postinoculation (H&E-stained section; original magnification ×20). Gross (M) and microscopic (N) pathology scores of monkeys infected with wild-type (red circles) or Δpvl (blue triangles) strains of USA300 were not significantly different at any single time point (Mann–Whitney test, P = not significant) or across all time points (one-way analysis of variance, P = not significant).

Figure 2

Microscopic examination of infected cynomolgus macaque lungs. Commonly observed features in animals infected with wild-type or Δpvl strains of USA300 included mild to moderate pneumonia with increased inflammatory cell infiltrates, predominately PMNs and monocytes, in the interstitium (boxed region) and alveolar space (arrows; A), bronchiolitis (B), vasculitis (C), capillary vascular congestion with microhemorrhage (arrow; D), pneumocyte hyperplasia (circled region) with desquamation (E), and macrophages with abundant foamy cytoplasm (F; H&E-stained section; original magnification ×40 [A–C] or ×20 [D–F]).

Figure 3

Perivascular cuffing of the small-to-medium sized arteries by induced bronchus-associated lymphoid tissue (IBALT). IBALT was observed in all animals at all time points, with three representative examples shown (A–C; boxed region) (H&E-stained section; original magnification ×40). The lymphocytic structures consisted of many CD3+ T-cells (arrows; D), scattered CD20+ B-cells (arrows; E), and lacked a well-organized CD21+ dendritic cell meshwork (absence of positively staining cells; F; Consecutive sections cut from the same tissue block were stained with the ABC immunostain technique, DAB chromagen; original magnification ×40).

Figure 4

Cytokines and acute phase reactants were measured in the serum and tissue of infected cynomolgus macaques. Similar levels of serum C-reactive protein (A), serum haptoglobin (B), serum myeloperoxidase (C), and lung tissue myeloperoxidase (D) were found in animals inoculated with wild-type (red bars) or Δpvl (blue bars) strains of USA300 (Mann–Whitney test, P = not significant). The near-baseline levels of serum C-reactive protein and myeloperoxidase at 8-day postinoculation may be consistent with histological evidence of early infection resolution.