Additional file 1 of Neutralization of the Staphylococcus aureus Panton-Valentine leukocidin by African and Caucasian sera

Additional file 1: Supplementary Fig. S1. Correlation of antibodies against Panton-Valentine leukocidin (PVL) with the neutralizing effect on PVL-induced cell damage. Serum levels of anti-PVL-antibodies are plotted against the amount of undamaged polymorphonuclear leukocytes (PMNs) from the African or German donor after treatment with 5 nM recombinant PVL in the presence of 0.625% or 2.5% serum from African (blue triangles) or Caucasian (orange circles) participants. Linear regression and correlation analyses of a given population (color-coding) are indicated as the coefficient of determination (R2) and Spearman’s correlation test coefficients (r) with probability (p), respectively

Sigma Factor SigB Is Crucial to Mediate <i>Staphylococcus aureus</i> Adaptation during Chronic Infections

<div><p><i>Staphylococcus aureus</i> is a major human pathogen that causes a range of infections from acute invasive to chronic and difficult-to-treat. Infection strategies associated with persisting <i>S</i>. <i>aureus</i> infections are bacterial host cell invasion and the bacterial ability to dynamically change phenotypes from the aggressive wild-type to small colony variants (SCVs), which are adapted for intracellular long-term persistence. The underlying mechanisms of the bacterial switching and adaptation mechanisms appear to be very dynamic, but are largely unknown. Here, we analyzed the role and the crosstalk of the global <i>S</i>. <i>aureus</i> regulators <i>agr</i>, <i>sarA</i> and SigB by generating single, double and triple mutants, and testing them with proteome analysis and in different <i>in vitro</i> and <i>in vivo</i> infection models. We were able to demonstrate that SigB is the crucial factor for adaptation in chronic infections. During acute infection, the bacteria require the simultaneous action of the <i>agr</i> and <i>sarA</i> loci to defend against invading immune cells by causing inflammation and cytotoxicity and to escape from phagosomes in their host cells that enable them to settle an infection at high bacterial density. To persist intracellularly the bacteria subsequently need to silence <i>agr</i> and <i>sarA</i>. Indeed <i>agr</i> and <i>sarA</i> deletion mutants expressed a much lower number of virulence factors and could persist at high numbers intracellularly. SigB plays a crucial function to promote bacterial intracellular persistence. In fact, Δ<i>sigB</i>-mutants did not generate SCVs and were completely cleared by the host cells within a few days. In this study we identified SigB as an essential factor that enables the bacteria to switch from the highly aggressive phenotype that settles an acute infection to a silent SCV-phenotype that allows for long-term intracellular persistence. Consequently, the SigB-operon represents a possible target to develop preventive and therapeutic strategies against chronic and therapy-refractory infections.</p></div

Differential expression of the regulators <i>agr</i>, <i>sarA</i> and <i>sigB</i> during the course of host cell infection in wild-type LS1 and the corresponding mutants.

<p>Endothelial cells were infected with <i>S</i>. <i>aureus</i> strain LS1 or the corresponding mutants as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004870#ppat.1004870.g003" target="_blank">Fig 3A</a> and infected cells were analysed for up to 7 days. Host cells infected with wild-type strain LS1 were lysed after 2 (acute phase) and 7 days (chronic phase) and the whole RNA was extracted and was used to determine changes in bacterial gene expression for <i>agrA/hla</i> (α-hemolysin) (A), <i>sarA/aur</i> (aureolysin) (B) and <i>sigB/asp23</i> (C) during the course of infection by real-time PCR. The values of all experiments represent the mean ± SD of 5 independent experiments measured in triplicate. * P≤0.05 ANOVA comparing levels of gene expression in the wild-type strains and the corresponding mutants at each time point. The fold change is the result of normalized expression to the housekeeping genes <i>gyr</i>, <i>aroE</i> and <i>gmk</i>. Uninfected cells were used as controls (Control = 1).</p

The interplay of <i>agr</i>, <i>sarA</i> and <i>sigB</i> is required to settle and maintain an infection in a local rat chronic osteomyelitis model.

<p>A local chronic osteomyelitis model was used to study the effects of wild-type SH1000 and corresponding mutants. (A) After 4 days of infection slices of bone tissue were performed for histology and stained by hematoxylin-eosin to detect the influx of immune cells to bone tissue. For each strain tested representative photomicrographs are shown in low and high magnification and typical histological features are described. (B, C) Bacterial persistence in host tissue was analyzed 4 days p.i. (acute) and 14 weeks p.i. (chronic) by plating homogenized bone tissue on agar plates and counting the CFU the following day. (D, E) The osteomyelitis index was measured 4 days p.i. (acute) and 14 weeks p.i. (chronic) from each infected tibiae in comparison with the non-infected tibiae from the same animal. The experiments were performed with 12 animals per group and the results shown are means ± SD. * P≤0.05 ANOVA test comparing persisting CFU and osteomyelitis index caused by wild-type SH1000 and the corresponding mutants. (F) Photographs of infected (wild-type SH1000) and non-infected tibiae recovered after 14 weeks.</p