Lisa E. Myers

Mn2+-Dependent Regulation of Multiple Genes in Streptococcus pneumoniae through PsaR and the Resultant Impact on Virulence

ABSTRACT The concentration of Mn2+ is 1,000-fold higher in secretions than it is at internal sites of the body, making it a potential signal by which bacteria can sense a shift from a mucosal environment to a more invasive site. PsaR, a metal-dependent regulator in Streptococcus pneumoniae, was found to negatively affect the transcription of psaBCA, pcpA, rrgA, rrgB, rrgC, srtBCD, and rlrA in the presence of Mn2+. psaBCA encode an ABC-type transporter for Mn2+. pcpA, rrgA, rrgB, and rrgC encode several outer surface proteins. srtBCD encode a cluster of sortase enzymes, and rlrA encodes a transcriptional regulator. Steady-state RNA levels are high under low Mn2+ concentrations in the wild-type strain and are elevated under both high and low Mn2+ concentrations in a psaR mutant strain. RlrA is an activator of rrgA, rrgB, rrgC, and srtBCD (D. Hava and A. Camilli, Mol. Microbiol. 45:1389-1406, 2002), suggesting that PsaR may indirectly control these genes through rlrA, while PsaR-dependent repression of psaBCA, pcpA, and rlrA transcription is direct. The impact of Mn2+-dependent regulation on virulence was further examined in mouse models of pneumonia and nasopharyngeal carriage. The abilities of ΔpsaR, pcpA, and ΔpsaR ΔpcpA mutant strains to colonize the lung were reduced compared to those of the wild type, confirming that both PcpA-mediated gene regulation and PsaR-mediated gene regulation are required for full virulence in the establishment of pneumonia. Neither PcpA nor PsaR was found to be required for colonization of the nasopharynx in a carriage model. This is the first demonstration of Mn2+ acting as a signal for the expression of virulence factors within different host sites.

Lipoprotein PsaA in Virulence of Streptococcus pneumoniae: Surface Accessibility and Role in Protection from Superoxide

ABSTRACT PsaA of Streptococcus pneumoniae, originally believed to be an adhesin, is the lipoprotein component of an Mn2+ transporter. Mutations in psaA cause deficiencies in growth, virulence, adherence, and the oxidative stress response. Immunofluorescence microscopy shows that PsaA is hidden beneath the cell wall and the polysaccharide capsule and only exposed to antibodies upon cell wall removal. A psaBC deletion mutant, expressing PsaA normally, was as deficient in adherence to Detroit 562 cells as were strains lacking PsaA. Thus, PsaA does not appear to act directly as an adhesin, but rather, psaA mutations indirectly affect this process through the disruption of Mn2+ transport. The deficiency in Mn2+ transport also causes hypersensitivity to oxidative stress from H2O2 and superoxide. In a chemically defined medium, growth of the wild-type strain was possible in the absence of Fe2+ and Mn2+ cations after a lag of about 15 h. Addition of Mn2+ alone or together with Fe2+ allowed prompt and rapid growth. In the absence of Mn2+, the addition of Fe2+ alone extended the 15-h lag phase to 25 h. Thus, while Fe2+ adversely affects the transition from lag phase to log phase, perhaps through increasing oxidative stress, this effect is relieved by the presence of Mn2+. A scavenger specific for superoxides but not those specific for hydroxyl radicals or H2O2 was able to eliminate the inhibition of growth caused by iron supplementation in the absence of Mn2+. This implies that superoxides are a key player in oxidative stress generated in the presence of iron.

Neutralizing Antibodies Elicited by a Novel Detoxified Pneumolysin Derivative, PlyD1, Provide Protection against Both Pneumococcal Infection and Lung Injury

ABSTRACT Streptococcus pneumoniae pneumolysin (PLY) is a virulence factor that causes toxic effects contributing to pneumococcal pneumonia. To date, deriving a PLY candidate vaccine with the appropriate detoxification and immune profile has been challenging. A pneumolysin protein that is appropriately detoxified and that retains its immunogenicity is a desirable vaccine candidate. In this study, we assessed the protective efficacy of our novel PlyD1 detoxified PLY variant and investigated its underlying mechanism of protection. Results have shown that PlyD1 immunization protected mice against lethal intranasal (i.n.) challenge with pneumococci and lung injury mediated by PLY challenge. Protection was associated with PlyD1-specific IgG titers and in vitro neutralization titers. Pretreatment of PLY with PlyD1-specific rat polyclonal antiserum prior to i.n. delivery of toxin reduced PLY-mediated lung lesions, interleukin-6 (IL-6) production, and neutrophil infiltration into lungs, indicating that protection from lung lesions induced by PLY is antibody mediated. Preincubation of PLY with a neutralizing monoclonal PLY antibody also specifically reduced the cytotoxic effects of PLY after i.n. inoculation in comparison to nonneutralizing monoclonal antibodies. These results indicate that the induction of neutralizing antibodies against PLY can contribute to protection against bacterial pneumonia by preventing the development of PLY-induced lung lesions and inflammation. Our detoxified PlyD1 antigen elicits such PLY neutralizing antibodies, thus serving as a candidate vaccine antigen for the prevention of pneumococcal pneumonia.

PcpA Promotes Higher Levels of Infection and Modulates Recruitment of Myeloid-Derived Suppressor Cells during Pneumococcal Pneumonia

We used two different infection models to investigate the kinetics of the PcpA-dependent pneumococcal disease in mice. In a bacteremic pneumonia model, we observed a PcpA-dependent increase in bacterial burden in the lungs, blood, liver, bronchoalveolar lavage, and spleens of mice at 24 h postinfection. This PcpA-dependent effect on bacterial burden appeared earlier (within 12 h) in the focal pneumonia model, which lacks bacteremia or sepsis. Histological changes show that the ability of pneumococci to make PcpA was associated with unresolved inflammation in both models of infection. Using our bacteremic pneumonia model we further investigated the effects of PcpA on recruitment of innate immune regulatory cells. The presence of PcpA was associated with increased IL-6 levels, suppressed production of TRAIL, and reduced infiltration of polymorphonuclear cells. The ability of pneumococci to make PcpA negatively modulated both the infiltration and apoptosis of macrophages and the recruitment of myeloid-derived suppressor-like cells. The latter have been shown to facilitate the clearance and control of bacterial pneumonia. Taken together, the ability to make PcpA was strongly associated with increased bacterial burden, inflammation, and negative regulation of innate immune cell recruitment to the lung tissue during bacteremic pneumonia.