EspZ of enteropathogenic and enterohemorrhagic Escherichia coli regulates type III secretion system protein translocation

UNLABELLED: Translocation of effector proteins via a type III secretion system (T3SS) is a widespread infection strategy among Gram-negative bacterial pathogens. Each pathogen translocates a particular set of effectors that subvert cell signaling in a way that suits its particular infection cycle. However, as effector unbalance might lead to cytotoxicity, the pathogens must employ mechanisms that regulate the intracellular effector concentration. We present evidence that the effector EspZ controls T3SS effector translocation from enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli. Consistently, an EPEC espZ mutant is highly cytotoxic. Following ectopic expression, we found that EspZ inhibited the formation of actin pedestals as it blocked the translocation of Tir, as well as other effectors, including Map and EspF. Moreover, during infection EspZ inhibited effector translocation following superinfection. Importantly, while EspZ of EHEC O157:H7 had a universal "translocation stop" activity, EspZ of EPEC inhibited effector translocation from typical EPEC strains but not from EHEC O157:H7 or its progenitor, atypical EPEC O55:H7. We found that the N and C termini of EspZ, which contains two transmembrane domains, face the cytosolic leaflet of the plasma membrane at the site of bacterial attachment, while the extracellular loop of EspZ is responsible for its strain-specific activity. These results show that EPEC and EHEC acquired a sophisticated mechanism to regulate the effector translocation.

IMPORTANCE: Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are important diarrheal pathogens responsible for significant morbidity and mortality in developing countries and the developed world, respectively. The virulence strategy of EPEC and EHEC revolves around a conserved type III secretion system (T3SS), which translocates bacterial proteins known as effectors directly into host cells. Previous studies have shown that when cells are infected in two waves with EPEC, the first wave inhibits effector translocation by the second wave in a T3SS-dependent manner, although the factor involved was not known. Importantly, we identified EspZ as the effector responsible for blocking protein translocation following a secondary EPEC infection. Interestingly, we found that while EspZ of EHEC can block protein translocation from both EPEC and EHEC strains, EPEC EspZ cannot block translocation from EHEC. These studies show that EPEC and EHEC employ a novel infection strategy to regulate T3SS translocation.

Legionella pneumophila secretes a mitochondrial carrier protein during infection

The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionellanucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms.

The enteropathogenic Escherichia coli effector NleH inhibits apoptosis induced by Clostridium difficile toxin B

Clostridium difficile is a leading cause of nosocomial infections, causing a spectrum of diseases ranging from diarrhoea to pseudomembranous colitis triggered by a range of virulence factors including C. difficile toxins A (TcdA) and B (TcdB). TcdA and TcdB are monoglucosyltransferases that irreversibly glycosylate small Rho GTPases, inhibiting their ability to interact with their effectors, guanine nucleotide exchange factors, and membrane partners, leading to disruption of downstream signalling pathways and cell death. In addition, TcdB targets the mitochondria, inducing the intrinsic apoptotic pathway resulting in TcdB-mediated apoptosis. Modulation of apoptosis is a common strategy used by infectious agents. Recently, we have shown that the enteropathogenic Escherichia coli (EPEC) type III secretion system effector NleH has a broad-range anti-apoptotic activity. In this study we examined the effects of NleH on cells challenged with TcdB. During infection with wild-type EPEC, NleH inhibited TcdB-induced apoptosis at both low and high toxin concentrations. Transfected nleH1 alone was sufficient to block TcdB-induced cell rounding, nuclear condensation, mitochondrial swelling and lysis, and activation of caspase-3. These results show that NleH acts via a global anti-apoptotic pathway.

Subcellular localization of legionella Dot/Icm effectors

The translocation of effector proteins by the Dot/Icm type IV secretion system is central to the ability of Legionella pneumophila to persist and replicate within eukaryotic cells. The subcellular localization of translocated Dot/Icm proteins in host cells provides insight into their function. Through co-staining with host cell markers, effector proteins may be localized to specific subcellular compartments and membranes, which frequently reflects their host cell target and mechanism of action. In this chapter, we describe protocols to (1) localize effector proteins within cells by ectopic expression using green fluorescent protein fusions and (2) localize effector proteins within infected cells using epitope-tagged effector proteins and immuno-fluorescence microscopy.