The opportunistic human pathogen Staphylococcus aureus causes serious infectious diseases that range from superficial skin and soft tissue infections to necrotizing pneumonia and sepsis. While classically regarded as an extracellular pathogen, S. aureus is able to invade and survive within human cells. Host cell exit is associated with cell death, tissue destruction, and the spread of infection. The exact molecular mechanism employed by S. aureus to escape the host cell is still unclear. In this study, we performed a genome-wide small hairpin RNA (shRNA) screen and identified the calcium signaling pathway as being involved in intracellular infection. S. aureus induced a massive cytosolic Ca\(^{2+}\) increase in epithelial host cells after invasion and intracellular replication of the pathogen. This was paralleled by a decrease in endoplasmic reticulum Ca\(^{2+}\) concentration. Additionally, calcium ions from the extracellular space contributed to the cytosolic Ca2+ increase. As a consequence, we observed that the cytoplasmic Ca\(^{2+}\) rise led to an increase in mitochondrial Ca\(^{2+}\) concentration, the activation of calpains and caspases, and eventually to cell lysis of S. aureus-infected cells. Our study therefore suggests that intracellular S. aureus disturbs the host cell Ca\(^{2+}\) homeostasis and induces cytoplasmic Ca\(^{2+}\) overload, which results in both apoptotic and necrotic cell death in parallel or succession. IMPORTANCE Despite being regarded as an extracellular bacterium, the pathogen Staphylococcus aureus can invade and survive within human cells. The intracellular niche is considered a hideout from the host immune system and antibiotic treatment and allows bacterial proliferation. Subsequently, the intracellular bacterium induces host cell death, which may facilitate the spread of infection and tissue destruction. So far, host cell factors exploited by intracellular S. aureus to promote cell death are only poorly characterized. We performed a genome-wide screen and found the calcium signaling pathway to play a role in S. aureus invasion and cytotoxicity. The intracellular bacterium induces a cytoplasmic and mitochondrial Ca\(^{2+}\) overload, which results in host cell death. Thus, this study first showed how an intracellular bacterium perturbs the host cell Ca\(^{2+}\) homeostasis."

Many pathogenic bacteria cause local infections but occasionally invade into the blood stream, often with fatal outcome. Very little is known about the mechanism underlying the switch from local to invasive infection. In the case of Neisseria gonorrhoeae, phase variable type 4 pili (T4P) stabilize local infection by mediating microcolony formation and inducing anti-invasive signals. Outer membrane porin PorBIA, in contrast, is associated with disseminated infection and facilitates the efficient invasion of gonococci into host cells. Here we demonstrate that loss of pili by natural pilus phase variation is a prerequisite for the transition from local to invasive infection. Unexpectedly, both T4P-mediated inhibition of invasion and PorBIA-triggered invasion utilize membrane rafts and signaling pathways that depend on caveolin-1-Y14 phosphorylation (Cav1-pY14). We identified p85 regulatory subunit of PI3 kinase (PI3K) and phospholipase Cγ1 as new, exclusive and essential interaction partners for Cav1-pY14 in the course of PorBIA-induced invasion. Active PI3K induces the uptake of gonococci via a new invasion pathway involving protein kinase D1. Our data describe a novel route of bacterial entry into epithelial cells and offer the first mechanistic insight into the switch from local to invasive gonococcal infection.

Metabolic adaptation to the host cell is important for obligate intracellular pathogens such as Chlamydia trachomatis (Ct). Here we infer the flux differences for Ct from proteome and qRT-PCR data by comprehensive pathway modeling. We compare the comparatively inert infectious elementary body (EB) and the active replicative reticulate body (RB) systematically using a genome-scale metabolic model with 321 metabolites and 277 reactions. This did yield 84 extreme pathways based on a published proteomics dataset at three different time points of infection. Validation of predictions was done by quantitative RT-PCR of enzyme mRNA expression at three time points. Ct’s major active pathways are glycolysis, gluconeogenesis, glycerol-phospholipid (GPL) biosynthesis (support from host acetyl-CoA) and pentose phosphate pathway (PPP), while its incomplete TCA and fatty acid biosynthesis are less active. The modeled metabolic pathways are much more active in RB than in EB. Our in silico model suggests that EB and RB utilize folate to generate NAD(P)H using independent pathways. The only low metabolic flux inferred for EB involves mainly carbohydrate metabolism. RB utilizes energy -rich compounds to generate ATP in nucleic acid metabolism. Validation data for the modeling include proteomics experiments (model basis) as well as qRT-PCR confirmation of selected metabolic enzyme mRNA expression differences. The metabolic modeling is made fully available here. Its detailed insights and models on Ct metabolic adaptations during infection are a useful modeling basis for future studies.

Staphylococcus aureus is a major human pathogen, which can invade and survive in non-professional and professional phagocytes. Uptake by host cells is thought to contribute to pathogenicity and persistence of the bacterium. Upon internalization by epithelial cells, cytotoxic S. aureus strains can escape from the phagosome, replicate in the cytosol and induce host cell death. Here, we identified a staphylococcal cysteine protease to induce cell death after translocation of intracellular S. aureus into the host cell cytoplasm. We demonstrated that loss of staphopain A function leads to delayed onset of host cell death and prolonged intracellular replication of S. aureus in epithelial cells. Overexpression of staphopain A in a non-cytotoxic strain facilitated intracellular killing of the host cell even in the absence of detectable intracellular replication. Moreover, staphopain A contributed to efficient colonization of the lung in a mouse pneumonia model. In phagocytic cells, where intracellular S. aureus is exclusively localized in the phagosome, staphopain A did not contribute to cytotoxicity. Our study suggests that staphopain A is utilized by S. aureus to exit the epithelial host cell and thus contributes to tissue destruction and dissemination of infection. Author summary Staphylococcus aureus is an antibiotic-resistant pathogen that emerges in hospital and community settings and can cause a variety of diseases ranging from skin abscesses to lung inflammation and blood poisoning. The bacterium can asymptomatically colonize the upper respiratory tract and skin of humans and take advantage of opportune conditions, like immunodeficiency or breached barriers, to cause infection. Although S. aureus was not regarded as intracellular bacterium, it can be internalized by human cells and subsequently exit the host cells by induction of cell death, which is considered to cause tissue destruction and spread of infection. The bacterial virulence factors and underlying molecular mechanisms involved in the intracellular lifestyle of S. aureus remain largely unknown. We identified a bacterial cysteine protease to contribute to host cell death of epithelial cells mediated by intracellular S. aureus. Staphopain A induced killing of the host cell after translocation of the pathogen into the cell cytosol, while bacterial proliferation was not required. Further, the protease enhanced survival of the pathogen during lung infection. These findings reveal a novel, intracellular role for the bacterial protease staphopain A.

Persistence has evolved as a potent survival strategy to overcome adverse environmental conditions. This capability is common to almost all bacteria, including all human bacterial pathogens and likely connected to chronic infections caused by some of these pathogens. Although the majority of a bacterial cell population will be killed by the particular stressors, like antibiotics, oxygen and nitrogen radicals, nutrient starvation and others, a varying subpopulation (termed persisters) will withstand the stress situation and will be able to revive once the stress is removed. Several factors and pathways have been identified in the past that apparently favor the formation of persistence, such as various toxin/antitoxin modules or stringent response together with the alarmone (p)ppGpp. However, persistence can occur stochastically in few cells even of stress-free bacterial populations. Growth of these cells could then be induced by the stress conditions. In this review, we focus on the persister formation of human intracellular bacterial pathogens, some of which belong to the most successful persister producers but lack some or even all of the assumed persistence-triggering factors and pathways. We propose a mechanism for the persister formation of these bacterial pathogens which is based on their specific intracellular bipartite metabolism. We postulate that this mode of metabolism ultimately leads, under certain starvation conditions, to the stalling of DNA replication initiation which may be causative for the persister state.