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Obstacles to bacterial survival and replication in the cytosol of host cells, and the mechanisms used by bacterial pathogens to adapt to this niche are not well understood. Listeria monocytogenes is a well-studied Gram-positive foodborne pathogen that has evolved to invade and replicate within the host cell cytosol; yet the mechanisms by which it senses and responds to stress to survive in the cytosol are largely unknown. To assess the role of the L. monocytogenes penicillin-binding-protein and serine/threonine associated (PASTA) kinase PrkA in stress responses, cytosolic survival and virulence, we constructed a ΔprkA deletion mutant. PrkA was required for resistance to cell wall stress, growth on cytosolic carbon sources, intracellular replication, cytosolic survival, inflammasome avoidance and ultimately virulence in a murine model of Listeriosis. In Bacillus subtilis and Mycobacterium tuberculosis, homologues of PrkA phosphorylate a highly conserved protein of unknown function, YvcK. We found that, similar to PrkA, YvcK is also required for cell wall stress responses, metabolism of glycerol, cytosolic survival, inflammasome avoidance and virulence. We further demonstrate that similar to other organisms, YvcK is directly phosphorylated by PrkA, although the specific site(s) of phosphorylation are not highly conserved. Finally, analysis of phosphoablative and phosphomimetic mutants of YvcK in vitro and in vivo demonstrate that while phosphorylation of YvcK is irrelevant to metabolism and cell wall stress responses, surprisingly, a phosphomimetic, nonreversible negative charge of YvcK is detrimental to cytosolic survival and virulence in vivo. Taken together our data identify two novel virulence factors essential for cytosolic survival and virulence of L. monocytogenes. Furthermore, our data demonstrate that regulation of YvcK phosphorylation is tightly controlled and is critical for virulence. Finally, our data suggest that yet to be identified substrates of PrkA are essential for cytosolic survival and virulence of L. monocytogenes and illustrate the importance of studying protein phosphorylation in the context of infection., Author Summary Infection with intracellular pathogens causes a majority of the global infectious disease associated mortality. A number of intracellular pathogens must directly access the host cytosol in order to cause disease; however, non-cytosol adapted bacteria do not survive or replicate upon access to the cytosol. The mechanisms cytosolic pathogens use to adapt to this niche are largely unknown. The model cytosolic bacterial pathogen Listeria monocytogenes contains a single penicillin-binding-protein and serine/threonine associated (PASTA) kinase, PrkA. In other bacteria, PASTA kinases bind cell wall fragments and phosphorylate downstream effectors involved in cell wall synthesis, central metabolism, virulence, cell division, and biofilm formation. We demonstrate that in L. monocytogenes, PrkA is required for cell wall homeostasis, growth under nutrient limiting conditions, survival and replication in host cells, and virulence in vivo. Furthermore, we identify a highly conserved protein of unknown function, YvcK, as a PrkA substrate. We demonstrate that L. monocytogenes YvcK is similarly required for cell wall stress responses, growth on glycerol, cytosolic survival and virulence in vivo. Surprisingly, a phosphomimetic, nonreversible negative charge at the phosphorylation sites on YvcK inactivates functions of the protein related to intracellular survival and virulence, suggesting that the identification of PASTA kinase substrates phosphorylated during infection will be critical to our understanding of this central regulator metabolism, cell wall homeostasis and ultimately virulence.

Certain intracellular bacteria use the host cell cytosol as the replicative niche. Although it has been hypothesized that the successful exploitation of this compartment requires a unique metabolic adaptation, supportive evidence is lacking. For Francisella tularensis , many genes of the Francisella pathogenicity island (FPI) are essential for intracellular growth, and therefore, FPI mutants are useful tools for understanding the prerequisites of intracytosolic replication. We compared the growth of bacteria taken up by phagocytic or nonphagocytic cells with that of bacteria microinjected directly into the host cytosol, using the live vaccine strain (LVS) of F. tularensis ; five selected FPI mutants thereof, i.e., Δ iglA , Δ iglC ̧ Δ iglG , Δ iglI , and Δ pdpE strains; and Listeria monocytogenes . After uptake in bone marrow-derived macrophages (BMDM), ASC −/− BMDM, MyD88 −/− BMDM, J774 cells, or HeLa cells, LVS, Δ pdpE and Δ iglG mutants, and L. monocytogenes replicated efficiently in all five cell types, whereas the Δ iglA and Δ iglC mutants showed no replication. After microinjection, all 7 strains showed effective replication in J774 macrophages, ASC −/− BMDM, and HeLa cells. In contrast to the rapid replication in other cell types, L. monocytogenes showed no replication in MyD88 −/− BMDM and LVS showed no replication in either BMDM or MyD88 −/− BMDM after microinjection. Our data suggest that the mechanisms of bacterial uptake as well as the permissiveness of the cytosolic compartment per se are important factors for the intracytosolic replication. Notably, none of the investigated FPI proteins was found to be essential for intracytosolic replication after microinjection.

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

The facultative intracellular pathogen Salmonella enterica resides in a specific membrane-bound compartment termed the Salmonella -containing vacuole (SCV). Despite being segregated from access to metabolites in the host cell cytosol, Salmonella is able to efficiently proliferate within the SCV. We set out to unravel the nutritional supply of Salmonella in the SCV with focus on amino acids. We studied the availability of amino acids by the generation of auxotrophic strains for alanine, asparagine, aspartate, glutamine, and proline in a macrophage cell line (RAW264.7) and an epithelial cell line (HeLa) and examined access to extracellular nutrients for nutrition. Auxotrophies for alanine, asparagine, or proline attenuated intracellular replication in HeLa cells, while aspartate, asparagine, or proline auxotrophies attenuated intracellular replication in RAW264.7 macrophages. The different patterns of intracellular attenuation of alanine- or aspartate-auxotrophic strains support distinct nutritional conditions in HeLa cells and RAW264.7 macrophages. Supplementation of medium with individual amino acids restored the intracellular replication of mutant strains auxotrophic for asparagine, proline, or glutamine. Similarly, a mutant strain deficient in succinate dehydrogenase was complemented by the extracellular addition of succinate. Complementation of the intracellular replication of auxotrophic Salmonella by external amino acids was possible if bacteria were proficient in the induction of Salmonella -induced filaments (SIFs) but failed in a SIF-deficient background. We propose that the ability of intracellular Salmonella to redirect host cell vesicular transport provides access of amino acids to auxotrophic strains and, more generally, is essential to continuously supply bacteria within the SCV with nutrients.

The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen that relies on the regulated secretion and activity of a variety of proteins that sustain life within diverse environments. PrsA2 has recently been identified as a secreted peptidyl-prolyl cis / trans isomerase and chaperone that is dispensable for bacterial growth in broth culture but essential for L. monocytogenes virulence. Following host infection, PrsA2 contributes to the proper folding and activity of secreted proteins that are required for bacterial replication within the host cytosol and for bacterial spread to adjacent cells. PrsA2 is one member of a family of Gram-positive secretion chaperones that appear to play important roles in bacterial physiology; however, it is not known how these proteins recognize their substrate proteins or the degree to which their function is conserved across diverse Gram-positive species. We therefore examined PrsA proteins encoded by a variety of Gram-positive bacteria for functional complementation of L. monocytogenes mutants lacking prsA2 . PrsA homologues encoded by Bacillus subtilis , Streptococcus pyogenes , Streptococcus pneumoniae , Streptococcus mutans , Staphylococcus aureus , and Lactococcus lactis were examined for functional complementation of a variety of L. monocytogenes PrsA2-associated phenotypes central to L. monocytogenes pathogenesis and bacterial cell physiology. Our results indicate that while selected aspects of PrsA2 function are broadly conserved among diverse Gram-positive bacteria, PrsA2 exhibits unique specificity for L. monocytogenes target proteins required for pathogenesis. The L. monocytogenes PrsA2 chaperone thus appears evolutionarily optimized for virulence factor secretion within the host cell cytosol while still maintaining aspects of activity relevant to more general features of Gram-positive protein translocation.

Listeria monocytogenes is a Gram-positive facultative intracellular bacterial pathogen that invades mammalian cells and escapes from membrane-bound vacuoles to replicate within the host cell cytosol. Gene products required for intracellular bacterial growth and bacterial spread to adjacent cells are regulated by a transcriptional activator known as PrfA. PrfA becomes activated following L. monocytogenes entry into host cells, however the signal that stimulates PrfA activation has not yet been defined. Here we provide evidence for L. monocytogenes secretion of a small peptide pheromone, pPplA, which enhances the escape of L. monocytogenes from host cell vacuoles and may facilitate PrfA activation. The pPplA pheromone is generated via the proteolytic processing of the PplA lipoprotein secretion signal peptide. While the PplA lipoprotein is dispensable for pathogenesis, bacteria lacking the pPplA pheromone are significantly attenuated for virulence in mice and have a reduced efficiency of bacterial escape from the vacuoles of nonprofessional phagocytic cells. Mutational activation of PrfA restores virulence and eliminates the need for pPplA-dependent signaling. Experimental evidence suggests that the pPplA peptide may help signal to L. monocytogenes its presence within the confines of the host cell vacuole, stimulating the expression of gene products that contribute to vacuole escape and facilitating PrfA activation to promote bacterial growth within the cytosol., Author Summary Experimental evidence has established that bacteria do not always exist as isolated single celled organisms, but instead communicate with each other through the secretion of small molecules that enable individual cells to coordinate complex traits and behaviors. Gram-positive bacteria rely on the secretion of small peptide pheromones to coordinate activities that include biofilm formation, exogenous DNA uptake via competence mechanisms, conjugal transfer of plasmid DNA, and expression of gene products that promote bacterial virulence. Here we provide evidence of a novel use of bacterial peptide pheromone signaling, that being the use of the pPplA peptide by L. monocytogenes to detect the confines of host cell vacuoles. Secretion and import of pPplA is required for efficient escape of L. monocytogenes from its initial membrane-bound host cell compartment, and bacteria lacking the peptide pheromone are severely attenuated for virulence in mice. The pPplA peptide pheromone is thus used by individual bacteria within a confined membrane bound space to coordinate the expression of gene products required by L. monocytogenes for intracellular growth and survival.

Chlamydia pneumoniae is an intracellular Gram-negative bacterium that possesses a type III secretion system (T3SS), which enables the pathogen to deliver, in a single step, effector proteins for modulation of host-cell functions into the human host cell cytosol to establish a unique intracellular niche for replication. The translocon proteins located at the top of the T3SS needle filament are essential for its function, as they form pores in the host-cell membrane. Interestingly, unlike other Gram-negative bacteria, C. pneumoniae has two putative translocon operons, named LcrH_1 and LcrH_2. However, little is known about chlamydial translocon proteins. In this study, we analyzed CPn0809, one of the putative hydrophobic translocators encoded by the LcrH_1 operon, and identified an 'SseC-like family' domain characteristic of T3S translocators. Using bright-field and confocal microscopy, we found that CPn0809 is associated with EBs during early and very late phases of a C. pneumoniae infection. Furthermore, CPn0809 forms oligomers, and interacts with the T3SS chaperone LcrH_1, via its N-terminal segment. Moreover, expression of full-length CPn0809 in the heterologous host Escherichia coli causes a grave cytotoxic effect that leads to cell death. Taken together, our data indicate that CPn0809 likely represents one of the translocon proteins of the C. pneumoniae T3SS, and possibly plays a role in the translocation of effector proteins in the early stages of infection.

The trimeric autotransporter adhesin Yersinia adhesin A is the prototype of the type Vc secretion systems. It is expressed by enteropathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis strains, but not by Yersinia pestis. A characteristic trait of YadA is its modular composition and trimeric nature. YadA consists of an N-terminal passenger domain which is exposed on the bacterial cell surface. The translocation of this passenger onto the surface is facilitated by a C-terminal β-barrel domain which concomitantly anchors YadA into the outer membrane with three YadA monomers contributing to the formation of a single β-barrel. In Y. enterocolitica, but not Y. pseudotuberculosis, YadA is a decisive virulence factor and its deletion renders the bacteria virtually avirulent in mouse models of infection. This striking importance of YadA in infection may derive from its manifold functions in host cell interaction. Presumably the most important function of YadA is that it mediates adhesion to extracellular matrix components of eukaryotic host cells. Only tight adhesion allows for the injection of "anti-host" effector proteins via a type III secretion system into the host cell cytosol. These effector proteins enable Yersinia to subvert the host immune system in order to replicate and establish infection. YadA is also essential for the survival of Y. enterocolitica upon contact with serum, an important immune-evasion mechanism called serum resistance. To this end, YadA interacts with several components of the host complement system, the first line of immune defense. This review will summarize recent findings about the structure and biogenesis of YadA and its interactions with the host complement system.

The intracellular bacterial pathogen Coxiella burnetii directs biogenesis of a parasitophorous vacuole (PV) that acquires host endolysosomal components. Formation of a PV that supports C. burnetii replication requires a Dot/Icm type 4B secretion system (T4BSS) that delivers bacterial effector proteins into the host cell cytosol. Thus, a subset of T4BSS effectors are presumed to direct PV biogenesis. Recently, the PV-localized effector protein CvpA was found to promote C. burnetii intracellular growth and PV expansion. We predict additional C. burnetii effectors localize to the PV membrane and regulate eukaryotic vesicle trafficking events that promote pathogen growth. To identify these vacuolar effector proteins, a list of predicted C. burnetii T4BSS substrates was compiled using bioinformatic criteria, such as the presence of eukaryote-like coiled-coil domains. Adenylate cyclase translocation assays revealed 13 proteins were secreted in a Dot/Icm-dependent fashion by C. burnetii during infection of human THP-1 macrophages. Four of the Dot/Icm substrates, termed C oxiella v acuolar p rotein B (CvpB), CvpC, CvpD, and CvpE, labeled the PV membrane and LAMP1-positive vesicles when ectopically expressed as fluorescently tagged fusion proteins. C. burnetii Δ cvpB , Δ cvpC , Δ cvpD , and Δ cvpE mutants exhibited significant defects in intracellular replication and PV formation. Genetic complementation of the Δ cvpD and Δ cvpE mutants rescued intracellular growth and PV generation, whereas the growth of C. burnetii Δ cvpB and Δ cvpC was rescued upon cohabitation with wild-type bacteria in a common PV. Collectively, these data indicate C. burnetii encodes multiple effector proteins that target the PV membrane and benefit pathogen replication in human macrophages.

Non-typhoidal Salmonella includes more than 2400 serotypes of Salmonella enterica that causes primarily enteric disease in human patients. Salmonella has a highly sophisticated molecular machinery that includes two type III secretion systems, which translocate bacterial effector proteins directly into the host cell cytosol. These systems subvert host cell pathways benefitting the pathogen, which ultimately allows for invasion of epithelial cells of the intestine, and intracellular survival and replication. These two critical steps in Salmonella pathogenesis are crucial for inducing intestinal inflammation, host colonization, and dissemination to systemic sites of infection. Salmonella triggers several somewhat overlapping pro-inflammatory pathways, which although they may play a favourable role mitigating systemic spread of infection, also favour the organism since Salmonella has evolved mechanisms that make it highly adapted to the inflamed intestinal environment. Thus, the Salmonella-elicited inflammatory response leads to competitive advantage of this organism over the intestinal microbiota.

Microbial pathogens and host immune cells each initiate events following their interaction in an attempt to drive the outcome to their respective advantage. Here we show that the bacterial pathogen Yersinia pseudotuberculosis sustains itself on the surface of a macrophage by forming acidic fluid-accessible compartments that are partially bounded by the host cell plasma membrane. These Yersinia-containing acidic compartments (YACs) are bereft of the early endosomal marker EEA1 and the lysosomal antigen LAMP1 and readily form on primary macrophages as well as macrophage-like cell lines. YAC formation requires the presence of the Yersinia virulence plasmid which encodes a type III secretion system. Unexpectedly, we found that the initial formation of YACs did not require translocation of the type III effectors into the host cell cytosol; however, the duration of YACs was markedly greater in infections using translocation-competent Y. pseudotuberculosis strains as well as strains expressing the effector YopJ. Furthermore, it was in this translocation- and YopJ-dependent phase of infection that the acidic environment was critical for Y. pseudotuberculosis survival during its interaction with macrophages. Our findings indicate that during its extracellular phase of infection Y. pseudotuberculosis initiates and then, by a separate mechanism, stabilizes the formation of a highly intricate structure on the surface of the macrophage that is disengaged from the endocytic pathway.

IFN-γ is critical for innate immunity against Listeria monocytogenes (L. monocytogenes), and it has long been thought that NK cells are the major source of IFN-γ during the first few days of infection. However, it was recently shown that a significant number of CD44highCD8+ T cells also secrete IFN-γ in an Ag-independent fashion within 16 h of infection with L. monocytogenes. In this report, we showed that infection with other intracellular pathogens did not trigger this early IFN-γ response and that cytosolic localization of Listeria was required to induce rapid IFN-γ production by CD44highCD8+ T cells. Infection of C57BL/6 mice with an Escherichia coli strain expressing listeriolysin O (LLO), a pore-forming toxin from L. monocytogenes, also resulted in rapid IFN-γ expression by CD8+ T cells. These results suggest that LLO expression is essential for induction of the early IFN-γ response, although it is not yet clear whether LLO plays a direct role in triggering a signal cascade that leads to cytokine production or whether it is required simply to release other bacterial product(s) into the host cell cytosol. Interestingly, mouse strains that displayed a rapid CD8+ T cell IFN-γ response (C57BL/6, 129, and NZB) all had lower bacterial burdens in the liver 3 days postinfection compared with mouse strains that did not have an early CD8+ T cell IFN-γ response (BALB/c, A/J, and SJL). These data suggest that participation of memory CD8+ T cells in the early immune response against L. monocytogenes correlates with innate host resistance to infection.

RIFINs are clonally variant antigens expressed in Plasmodium falciparum. Transfection and the green fluorescence protein (GFP) tagged either internally or C-terminally to the 3D7 PFI0050c RIFIN gene product were used to investigate protein localization, orientation and trafficking. Green fluorescence pattern emerging from live transfectant parasites expressing each of the RIFIN–GFP chimera was different. The internally GFP-tagged protein was exported to Maurer’s clefts (MC) in the erythrocyte cytosol, whereas the C-terminally GFP-tagged full-length RIFIN chimera was not trafficked out of the parasite. Interestingly, when some RIFIN-specific C-terminal amino acid sequences were removed, the resulting truncated molecule reached the MC. Using anti-RIFIN and anti-GFP antibodies to probe both live and fixed transfectants, staining was confined to MC and was not detected on the erythrocyte surface, a location previously suggested for this protein family. From selective permeabilization experiments, the highly variable portion of the RIFIN– GFP-insertion chimera appeared to be exposed to the erythrocyte cytosol, presumably anchored in the MC membrane via the two transmembrane domains. Trafficking of both chimeras in young ring stages was sensitive to Brefeldin A (BFA), although older rings showed differential sensitivity to BFA.

Strains of Listeria monocytogenes (LM) that are deficient in the virulence factor listeriolysin O (LLO) are highly attenuated and are thought not to elicit protective immunity. This failure has been attributed to the inability of the bacterium to enter the host cell cytosol and access MHC class I Ag processing machinery. We reexamined this issue using recombinant strains of LM that are deficient in LLO but express an additional CD8 T cell epitope derived from lymphocytic choriomeningitis virus. After infection with LLO-deficient strains, we find sizable priming of epitope-specific CD8 T cells and the development of a functional memory cell population. Mice primed with the LLO-deficient LM strain are equally resistant against high-dose challenge with virulent LM as mice primed with wild-type virulent bacteria and also resist heterologous challenge with lymphocytic choriomeningitis virus. Interestingly, priming with a low dose of LLO-deficient LM, which occurred in environment of reduced inflammation (IFN-γ), allowed rapid amplification of Ag-specific CD8 T cells by booster immunization, despite an undetectable primary response. We conclude that the generation of protective immunity by LLO-deficient strains of LM does in fact occur and that this highly attenuated LM strain may be a useful platform for vaccine delivery.

Environmental pathogens are organisms that normally spend a substantial part of their lifecycle outside of human hosts, but when introduced into humans are capable of causing disease. Such organisms are often able to transition between disparate environments ranging from the soil to the cytosol of host cells. The food-borne bacterial pathogen Listeria monocytogenes serves as a model system for understanding how an environmental organism makes the transition into mammalian hosts. A transcriptional regulatory protein known as PrfA appears to serve as a critical switch, enabling L. monocytogenes to transition from the outside environment to life within the host cell cytosol. PrfA is required for the expression of many L. monocytogenes gene products associated with virulence, and multiple mechanisms serve to regulate the expression and activity of PrfA. Increasing evidence suggests that specific environmental stresses help prime L. monocytogenes for life within the host, and cross-talk between the stress response regulator σ-B and PrfA may mediate the transition from outside environment to cytosol. Once within the host cytosol, multiple changes in bacterial metabolism and gene expression help to complete the transformation of L. monocytogenes from soil dweller to intracellular pathogen.

After invasion of erythrocytes, the human malaria parasite Plasmodium falciparum resides within a parasitophorous vacuole (PV) which forms an interface between the host cell cytosol and the parasite surface. This vacuole protects the parasite from potentially harmful substances, but allows access of essential nutrients to the parasite. Furthermore, the vacuole acts as a transit compartment for parasite proteins en route to the host cell cytoplasm. Recently we developed a strategy to biotin label soluble proteins of the PV. Here, we have paired this strategy with a high-throughput MALDI-TOF-MS analysis to identify 27 vacuolar proteins. These proteins fall into the following main classes: chaperones, proteases, and metabolic enzymes, consistent with the expected functions of the vacuole. These proteins are likely to be involved in several processes including nutrient acquisition from the host cytosol, protein sorting within the vacuole, and release of parasites at the end of the intraerythrocytic cycle.

The Gram-negative plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria translocates effector proteins via a specialized type III secretion (TTS) system into the host cell cytosol. The efficient secretion of many effector proteins depends on the global TTS chaperone HpaB. Here, we identified a novel export control protein, HpaC, which significantly contributes to bacterial pathogenicity. Deletion of hpaC leads to a severe reduction in secretion of effector proteins and the putative type III translocon proteins HrpF and XopA. By contrast, secretion of the TTS pilus protein HrpE is not affected. We provide experimental evidence that HpaC differentiates between two classes of effector proteins. Using an in vivo reporter assay, we found that HpaC specifically promotes the translocation of the effector proteins XopJ and XopF1 into the plant cell, whereas AvrBs3 and XopC are efficiently translocated even in the absence of HpaC. Similar findings were obtained for HpaB. Inhibition of protein synthesis suggests that HpaB is involved in the secretion of stored effector proteins. Furthermore, protein-protein interaction studies revealed that HpaB and HpaC form an oligomeric protein complex and that they interact with members of both effector protein classes and the conserved TTS system component HrcV. Taken together, our data indicate that HpaB and HpaC play a central role in recruiting TTS substrates to the secretion apparatus.

Two virulence factors ofListeria monocytogenes, listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC), mediate escape of this pathogen from the phagocytic vacuole of macrophages, thereby allowing the bacterium access to the host cell cytosol for growth and spread to neighboring cells. We characterized their orthologs fromBacillus anthracisby expressing them inL. monocytogenesand characterizing their contribution to bacterial intracellular growth and cell-to-cell spread. We generated a series ofL. monocytogenesstrains expressingB. anthracisanthrolysin O (ALO) and PI-PLC in place of LLO andL. monocytogenesPI-PLC, respectively. We found that ALO was active at both acidic and neutral pH and could functionally replace LLO in mediating escape from a primary vacuole; however, ALO exerted a toxic effect on the host cell by damaging the plasma membrane.B. anthracisPI-PLC, unlike theL. monocytogenesortholog, had high activity on glycosylphosphatidylinositol-anchored proteins.L. monocytogenesexpressingB. anthracisPI-PLC showed significantly decreased efficiencies of escape from a phagosome and in cell-to-cell spread. We further compared the level of cytotoxicity to host cells by using mutant strains expressing ALO in combination either withL. monocytogenesPI-PLC or withB. anthracisPI-PLC. The results demonstrated that the mutant strain expressing the combination of ALO andB. anthracisPI-PLC caused less damage to host cells than the strain expressing ALO andL. monocytogenesPI-PLC. The present study indicates that LLO andL. monocytogenesPI-PLC has adapted forL. monocytogenesintracellular growth and virulence and suggests that ALO andB. anthracisPI-PLC may have a role inB. anthracispathogenesis.

Listeria monocytogenes is a bacterial pathogen that elicits a strong cellular immune response and thus has potential use as a vaccine vector. An attenuated strain, L. monocytogenes dal dat , produced by deletion of two genes ( dal and dat ) used for d -alanine synthesis, induces cytotoxic T lymphocytes and protective immunity in mice following infection in the presence of d -alanine. In order to obviate the dependence of L. monocytogenes dal dat on supplemental d -alanine yet retain its attenuation and immunogenicity, we explored mechanisms to allow transient endogenous synthesis of the amino acid. Here, we report on a derivative strain, L. monocytogenes dal dat/ pRRR, that expresses a dal gene and synthesizes d -alanine under highly selective conditions. We constructed the suicide plasmid pRRR carrying a dal gene surrounded by two res1 sites and a resolvase gene, tnpR , which acts at the res1 sites. The resolvase gene is regulated by a promoter activated upon exposure to host cell cytosol. L. monocytogenes dal dat/ pRRR was thus able to grow in liquid culture and to infect host cells without d -alanine supplementation. However, after infection of these cells, resolvase-mediated excision of the dal gene resulted in strong down-regulation of racemase expression. As a result, this system allowed only transient growth of L. monocytogenes dal dat/ pRRR in infected cells and survival in animals for only 2 to 3 days. Nevertheless, mice immunized with L. monocytogenes dal dat/ pRRR generated listeriolysin O-specific effector and memory CD8 + T cells and were protected against lethal challenge by wild-type Listeria . This vector may be an attractive vaccine candidate for the induction of protective cellular immune responses.

Listeria monocytogenes is a facultative intracellular bacterial pathogen that causes serious disease in immunocompromised individuals, pregnant women, and neonates. Bacterial virulence is mediated by the expression of specific gene products that facilitate entry into host cells and enable bacterial replication; the majority of these gene products are regulated by a transcriptional activator known as PrfA. L. monocytogenes strains containing prfA E77K or prfA G155S mutations exhibit increased expression of virulence genes in broth culture and are hypervirulent in mice. To define the scope of the influences of the prfA E77K and prfA G155S mutations on L. monocytogenes pathogenesis, multiple aspects of bacterial invasion and intracellular growth were examined. Enhanced bacterial invasion of host epithelial cells was dependent on the expression of a number of surface proteins previously associated with invasion, including InlA, InlB, and ActA. In addition to these surface proteins, increased production of the hly -encoded secreted hemolysin listeriolysin O (LLO) was also found to significantly enhance bacterial invasion into epithelial cell lines for both prfA mutant strains. Although prfA E77K and prfA G155S strains were similar in their invasive phenotypes, the infection of epithelial cells with prfA E77K strains resulted in host cell plasma membrane damage, whereas prfA G155S strains did not alter plasma membrane integrity. Bacterial infection of human epithelial cells, in which the production of LLO is not required for bacterial entry into the cytosol, indicated that prfA E77K cytotoxic effects were mediated via LLO. Both prfA E77K and prfA G155S strains were more efficient than wild-type bacteria in gaining access to the host cell cytosol and in initiating the polymerization of host cell actin, and both were capable of mediating LLO-independent lysis of host cell vacuoles in cell lines for which L. monocytogenes vacuole disruption normally requires LLO activity. These experiments illuminate the diverse facets of L. monocytogenes pathogenesis that are significantly enhanced by the constitutive activation of PrfA via prfA mutations and underscore the critical role of this protein in promoting L. monocytogenes virulence.

The intracellular bacterial pathogen Chlamydia is sequestered from the host cell cytoplasm by remaining within an inclusion body during its replication cycle. Nevertheless, CD8+ T cells recognizing Chlamydia Ags in the context of MHC class I molecules are primed during infection. We have recently described derivation of Chlamydia-specific human CD8+ T cells by using infected dendritic cells as a surrogate system to reflect Chlamydia-specific CD8+ T cell responses in vivo. These CD8+ T cell clones recognize chlamydial Ags processed via the conventional class Ia processing pathway, as assessed by treatment of infected APC with lactacystin and brefeldin A, suggesting that the Ags are translocated from the chlamydial inclusion into the host cell cytosol. In this study, outer membrane protein 2 (OmcB) was identified as the Ag recognized by one of these Chlamydia-specific human CD8+ T cells, and we defined the HLA*A0101-restricted epitope from this Ag. CD8+ T cell responses to this epitope were present at high frequencies in the peripheral blood of both of two HLA*A0101 donors tested. In vitro chlamydial growth was completely inhibited by the OmcB-specific CD8+ T cell clone independently of lytic mechanisms. OmcB is a 60-kDa protein that has been postulated to be associated with the Chlamydia outer membrane complex. The subcellular localization of OmcB to the cytosol of infected cells, as determined by conventional MHC class I Ag processing and presentation, suggests the possibility of an additional, cytosolic-associated function for this protein.

The type III secretion system (TTSS) of plant- and animal-pathogenic bacteria is involved in translocation of virulence factors into the host cell cytosol where they modulate cellular processes. Sinorhizobium fredii USDA257 is a gram-negative soil bacterium that forms nitrogen-fixing nodules on specific soybean cultivars (Glycine max (L.) Merr.). This microsymbiont is known to secrete at least five nodulation outer proteins (Nops) in response to flavonoid induction. Some of these Nops have been shown to be secreted by TTSS in this symbiotic bacterium. We have isolated and purified an 18-kDa extracellular protein from flavonoid-induced cultures of USDA257. The N-terminal amino acid sequence of this purified protein was identical to the published sequence of the soybean cultivar-specificity protein, NopB (formerly NolB). Inactivation of rhcN, which encodes an ATPase, abolished secretion of NopB. Similarly, a nonpolar nopB deletion mutant was compromised in its ability to secrete several Nops. A construct containing the coding region of nopB under control of a T7 promoter was expressed successfully in Escherichia coli and, subsequently, the recombinant NopB was purified by nickel-affinity column chromatography. Polyclonal antibodies raised against purified recombinant NopB were used in Western blot analysis to demonstrate the association of NopB with pilus-like surface appendages. Deletion analysis indicated that the first 33 N-terminal residues of NopB were necessary and sufficient to mediate the secretion of a green fluorescent protein reporter. Introduction of plasmid-borne extra copies of nopB into USDA257 resulted in lower accumulation of native NopB. We also show that USDA257 and its nonpolar nopB deletion mutant exhibited discernible differences in their ability to nodulate legume hosts.

Mitochondria evolved from an endosymbiotic proteobacterium in a process that required the transfer of genes from the bacterium to the host cell nucleus, and the translocation of proteins thereby made in the host cell cytosol into the internal compartments of the organelle. According to current models for this evolution, two highly improbable events are required to occur simultaneously: creation of a protein translocation machinery to import proteins back into the endosymbiont and creation of targeting sequences on the protein substrates themselves. Using a combination of two independent prediction methods, validated through tests on simulated genomes, we show that at least 5% of proteins encoded by an extant proteobacterium are predisposed for targeting to mitochondria, and propose we that mitochondrial targeting information was preexisting for many proteins of the endosymbiont. We analyzed a family of proteins whose members exist both in bacteria and in mitochondria of eukaryotes and show that the amino-terminal extensions occasionally found in bacterial family members can function as a crude import sequence when the protein is presented to isolated mitochondria. This activity leaves the development of a primitive translocation channel in the outer membrane of the endosymbiont as a single hurdle to initiating the evolution of mitochondria.

The non-canonical inflammasome triggers host cell death and inflammation upon recognition of cytosolic LPS. A recent report in Cell now shows that Outer Membrane Vesicles (OMVs) of extracellular Gram-negative bacteria can deliver LPS into the host cell cytosol.

Listeria monocytogenesis a facultative intracellular bacterial pathogen that escapes from a phagosome and grows in the host cell cytosol. Escape of the bacterium from the phagosome to the cytosol is mediated by the bacterial pore-forming protein listeriolysin O (LLO). LLO has multiple mechanisms that optimize activity in the phagosome and minimize activity in the host cytosol. Mutants that fail to compartmentalize LLO activity are cytotoxic and have reduced virulence. We sought to determine why cytotoxic bacteria have attenuated virulence in the mouse model of listeriosis. In this study, we constructed a series of strains with mutations in LLO and with various degrees of cytotoxicity. We found that the more cytotoxic the strain in cell culture, the less virulent it was in mice. Induction of neutropenia increased the relative virulence of the cytotoxic strains 100-fold in the spleen and 10-fold in the liver. The virulence defect was partially restored in neutropenic mice by adding gentamicin, an antibiotic that kills extracellular bacteria. Additionally,L. monocytogenesgrew more slowly in extracellular fluid (mouse serum) than within tissue culture cells. We concluded thatL. monocytogenescontrols the cytolytic activity of LLO to maintain its nutritionally rich intracellular niche and avoid extracellular defenses of the host.

The asexual maturation of Plasmodium falciparum is accompanied by the transport of parasite-encoded proteins to the erythrocyte plasma membrane. Activation of G proteins by treatment with aluminum fluoride produced an accumulation within the erythrocyte cytosol of vesicles coated with Plasmodium homologues of COPII and N-ethylmaleimide-sensitive factor, proteins involved in intracellular transport between the Golgi apparatus and the endoplasmic reticulum. These vesicles contain malarial proteins that appear on the erythrocyte plasma membrane, as well as actin and myosin. It is proposed that the parasite adapted a process well established for intracellular transport to mediate the extracellular movement of its proteins through the erythrocyte cytosol to the surface membrane.

The facultative intracellular bacterial pathogen Listeria monocytogenes dramatically increases the expression of several key virulence factors upon entry into the host cell cytosol. actA, the protein product of which is required for cell-to-cell spread of the bacterium, is expressed at low to undetectable levels in vitro and increases in expression more than 200-fold after L. monocytogenes escape from the phagosome. To identify bacterial factors that participate in the intracellular induction of actA expression, L. monocytogenes mutants expressing high levels of actA during in vitro growth were selected after chemical mutagenesis. The resulting mutant isolates displayed a wide range of actA expression levels, and many were less sensitive to environmental signals that normally mediate repression of virulence gene expression. Several isolates contained mutations affecting actA gene expression that mapped at least 40 kb outside the PrfA regulon, supporting the existence of additional regulatory factors that contribute to virulence gene expression. Two actA in vitro expression mutants contained novel mutations within PrfA, a key regulator of L. monocytogenes virulence gene expression. PrfA E77K and PrfA G155S mutations resulted in high-level expression of PrfA-dependent genes, increased bacterial invasion of epithelial cells and increased virulence in mice. Both prfA mutant strains were significantly less motile than wild-type L. monocytogenes. These results suggest that, although constitutive activation of PrfA and PrfA-dependent gene expression may enhance L. monocytogenes virulence, it may conversely hamper the bacterium's ability to compete in environments outside host cells.

Nontyphoid Salmonella enterica requires the plasmid-encoded spv genes to establish successful systemic infection in experimental animals. The SpvB virulence-associated protein has recently been shown to contain the ADP-ribosyltransferase domain. SpvB ADP-ribosilates actin and depolymerizes actin filaments when expressed in cultured epithelial cells. However, spontaneous secretion or release of SpvB has not been observed under in vitro growth conditions. In the present study we investigated the secretion of SpvB from Salmonella using in vitro and in vivo assay systems. We showed that SpvB is secreted into supernatant from Salmonella strains that contain the cloned spvB gene on a plasmid when they grew in intracellular salts medium (ISM), a minimal medium mimicing the intracellular iron concentrations of eukaryotic cells. A series of mutant SpvB proteins revealed that an N-terminal region of SpvB located at amino acids 1-229 was sufficient to promote secretion into extracellular milieu. Confocal immunofluorescence microscopy also demonstrated efficient localization of the N-terminal domain of SpvB(1-360) tagged with biotinylated peptide within infected host cell cytosol but not truncated SpvB(1-179) fusion protein. In addition, mutations that inactivate genes within Salmonella pathogenicity island 1 or Salmonella pathogenicity island 2 that encode type III secretion systems (TTSS) could secrete the SpvB protein into the culture medium. These results indicate that SpvB protein is transported from the bacteria and into the host cytoplasm independent of TTSS.

The immune system has evolved various effector cells and functions to combat diverse infectious agents equipped with different virulence strategies. CD8 T cells play a critical role in protective immunity to Listeria monocytogenes (Lm), a bacterium that grows within the host cell cytosol and spreads directly into neighboring cells. The importance of CD8 T cells during Lm infection is currently attributed to the cytosolic niche of this organism, which allows it to evade many aspects of immune surveillance. CTL lysis of infected cells is believed to be an essential protective mechanism, presumably functioning to release intracellular bacteria, although its precise role remains to be fully defined. In this study, we examined the contribution of perforin-mediated CTL cytolysis to protective immunity against recombinant Lm capable of or defective in cell-cell spread. We found that CTL cytolysis is critical for protective immunity to Lm capable of cell-cell spread while protective immunity against spread-defective Lm is largely independent of CTL cytolysis. These results demonstrate that an important function of CTL cytolysis is to counter the microbial virulence strategy of direct cell-cell spread. We propose a model that advances the current view of the role of CTL cytolysis in immunity to intracellular pathogens.

Pathogenicity of most Gram-negative bacterial plant pathogens depends on hrp (hypersensitive response and pathogenicity) genes, which control the ability to cause disease and to elicit specific defense responses in resistant plants. hrp genes encode a specialized type III secretion (TTS) system that mediates the vectorial delivery of bacterial effector proteins across both bacterial membranes as well as across the eukaryotic plasma membrane into the host cell cytosol. One well-studied effector protein is AvrBs3 from Xanthomonas campestris pv. vesicatoria, the causal agent of bacterial spot in pepper and tomato. AvrBs3 induces hypertrophy symptoms in susceptible plants and triggers a resistance gene-specific cell death reaction in resistant plants. Intriguingly, AvrBs3 has characteristic features of eukaryotic transcription factors, suggesting that it modulates the host's transcriptome. Here, we discuss the TTS system of X.campestris pv. vesicatoria in the light of current knowledge on type III-dependent protein secretion in plant pathogenic bacteria.

In its host erythrocyte, the malaria parasite Plasmodium falciparum resides within a parasitophorous vacuole, the membrane of which forms a barrier between the host cell cytosol and the parasite surface. The vacuole is a unique compartment because it contains specific proteins that are believed to be involved in cell biological functions essential for parasite survival. As a prerequisite for the characterization of the vacuolar proteome, we have developed an experimental approach that allows the selective biotinylation of soluble vacuolar proteins. This approach utilizes nonpermeant biotin derivatives that can be introduced into infected erythrocytes after selective permeabilization of the erythrocyte membrane with the pore-forming protein streptolysin O. The derivatives gain access to the vacuolar lumen but not to the parasite cytosol, thus providing supportive evidence for the existence of nonselective pores within the vacuolar membrane that have been postulated based on electrophysiological studies. Soluble vacuolar proteins that are biotin-labeled can be isolated by affinity chromatography using streptavidin-agarose.

Intracellular pathogens have developed different mechanisms which enable their survival and replication within the host cells. Some survive and replicate within a membrane-bound vacuole modified by the bacteria to support microbial growth (e.g. Salmonella enterica serovar Typhimurium), whereas others escape from the vacuole into the host cell cytosol, where they proliferate (e.g. Listeria monocytogenes). In this study a Salmonella strain carrying a mutation in sifA which is released from the vacuole was used to analyse Salmonella survival and replication within the cytosol of several cell lines. It was found that Salmonella replicates within the cytosol of epithelial cells at a higher rate than that achieved when replicating within the vacuole, but is defective for replication in the cytosol of fibroblasts or macrophages. Using an aroC purD double mutant strain which does not replicate within host cells, it was shown that Salmonella encounters a killing activity within the cytosol of macrophages. Furthermore, in vitro experiments using cytosol extracted from either infected or uninfected macrophages suggested that this activity is activated upon Salmonella infection.

The bacterial enteropathogen Salmonella typhimurium employs a type III secretion system to inject bacterial toxins into the host cell cytosol. These toxins transiently activate Rho family GTP-binding protein-dependent signaling cascades to induce cytoskeletal rearrangements. One of these translocated Salmonella toxins, SopE, can activate Cdc42 in a Dbl-like fashion despite its lack of sequence similarity to Dbl-like proteins, the Rho-specific eukaryotic guanine nucleotide exchange factors. To elucidate the mechanism of SopE-mediated guanine nucleotide exchange, we have analyzed the structure of the complex between a catalytic fragment of SopE and Cdc42. SopE binds to and locks the switch I and switch II regions of Cdc42 in a conformation that promotes guanine nucleotide release. This conformation is strikingly similar to that of Rac1 in complex with the eukaryotic Dbl-like exchange factor Tiam1. However, the catalytic domain of SopE has an entirely different architecture from that of Tiam1 and interacts with the switch regions via different amino acids. Therefore, SopE represents the first example of a non-Dbl-like protein capable of inducing guanine nucleotide exchange in Rho family proteins.

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a phagosome and grows in the host cell cytosol. The pore-forming cholesterol-dependent cytolysin, listeriolysin O (LLO), mediates bacterial escape from vesicles and is approximately 10-fold more active at an acidic than neutral pH. By swapping dissimilar residues from a pH-insensitive orthologue, perfringolysin O (PFO), we identified leucine 461 as unique to pathogenic Listeria and responsible for the acidic pH optimum of LLO. Conversion of leucine 461 to the threonine present in PFO increased the hemolytic activity of LLO almost 10-fold at a neutral pH. L. monocytogenes synthesizing LLO L461T, expressed from its endogenous site on the bacterial chromosome, resulted in a 100-fold virulence defect in the mouse listeriosis model. These bacteria escaped from acidic phagosomes and initially grew normally in cells and spread cell to cell, but prematurely permeabilized the host membrane and killed the cell. These data show that the acidic pH optimum of LLO results from an adaptive mutation that acts to limit cytolytic activity to acidic vesicles and prevent damage in the host cytosol, a strategy also used by host cells to compartmentalize lysosomal hydrolases.

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a phagosome and grows in the host cell cytosol. The pore-forming cholesterol-dependent cytolysin, listeriolysin O (LLO), mediates bacterial escape from vesicles and is ∼10-fold more active at an acidic than neutral pH. By swapping dissimilar residues from a pH-insensitive orthologue, perfringolysin O (PFO), we identified leucine 461 as unique to pathogenic Listeria and responsible for the acidic pH optimum of LLO. Conversion of leucine 461 to the threonine present in PFO increased the hemolytic activity of LLO almost 10-fold at a neutral pH. L. monocytogenes synthesizing LLO L461T, expressed from its endogenous site on the bacterial chromosome, resulted in a 100-fold virulence defect in the mouse listeriosis model. These bacteria escaped from acidic phagosomes and initially grew normally in cells and spread cell to cell, but prematurely permeabilized the host membrane and killed the cell. These data show that the acidic pH optimum of LLO results from an adaptive mutation that acts to limit cytolytic activity to acidic vesicles and prevent damage in the host cytosol, a strategy also used by host cells to compartmentalize lysosomal hydrolases.

It is generally accepted that intraerythrocytic malaria parasites digest hemoglobin to supply the amino acids needed for the synthesis of their own proteins. This view has never been quantitatively tested. In this investigation we have measured the degradation of hemoglobin and the increase in parasite protein content as a function of parasite maturation in cultures of Plasmodium falciparum. Defined parasite stages were obtained either from tightly synchronized cultures or from asynchronous cultures after density-fractionation. We showed that both hemoglobin digestion and total parasite protein content increased with parasite maturation, from the early trophozoite stage onwards, although the total protein content of the parasite remained significantly lower than that of other eukaryotes. The parasite digested up to 65% of the host cell's hemoglobin but utilized only up to about 16% of the amino acids derived from hemoglobin digestion. This large discrepancy is profoundly puzzling particularly in view of the need to detoxify the cell from the large quantities of ferriprotoporphyrin IX and iron released during hemoglobin digestion.

Salmonella enterica is a bacterial pathogen of humans that can proliferate within epithelial cells as well as professional phagocytes of the immune system. This ability requires an S. enterica specific locus termed Salmonella pathogenicity island 2 (SPI-2). SPI-2 encodes a type III secretion system that injects effectors encoded within the island into host cell cytosol to promote virulence. SsrAB is a two-component regulator encoded within SPI-2 that was assumed to activate SPI-2 genes exclusively. Here, it is shown that SsrB in fact activates a global regulon. At least 10 genes outside SPI-2 are SsrB regulated within epithelial and macrophage cells. Nine of these 10 SsrB-regulated genes outside SPI-2 reside within previously undescribed regions of the Salmonella genome. Most share no sequence homology with current database entries. However, one is remarkably homologous to human glucosyl ceramidase, an enzyme involved in the ceramide signalling pathway. The SsrB regulon is modulated by the two-component regulatory systems PhoP/PhoQ and OmpR/EnvZ, and is upregulated in the intracellular microenvironment.

In many Gram-negative bacteria, a key indicator of pathogenic potential is the possession of a specialized type III secretion system, which is utilized to deliver virulence effector proteins directly into the host cell cytosol. Many of the proteins secreted from such systems require small cytosolic chaperones to maintain the secreted substrates in a secretion-competent state. One such protein, CesT, serves a chaperone function for the enteropathogenic Escherichia coli (EPEC) translocated intimin receptor (Tir) protein, which confers upon EPEC the ability to alter host cell morphology following intimate bacterial attachment. Using a combination of complementary biochemical approaches, functional domains of CesT that mediate intermolecular interactions, involved in both chaperone-chaperone and chaperone-substrate associations, were determined. The CesT N-terminal is implicated in chaperone dimerization, whereas the amphipathic alpha-helical region of the C-terminal, is intimately involved in substrate binding. By functional complementation of chaperone domains using the Salmonella SicA chaperone to generate chaperone chimeras, we show that CesT-Tir interaction proceeds by a mechanism potentially common to other type III secretion system chaperones.

The bacterial pathogen Listeria monocytogenes (Lm) is the causative agent of listeriosis, a rare but fatal foodborne disease. During infection, Lm can traverse several host barriers and enter the cytosol of a variety of cell types. Thus, consideration of the extracellular and intracellular niches of Lm is critical for understanding the infection process. Here, we review advances in our understanding of Lm infection and highlight how the interactions between the host and the pathogen are context dependent. We discuss discoveries of how Lm senses entry into the host cell cytosol. We present findings concerning how the nature of the various cytoskeleton components subverted by Lm changes depending on both the stage of infection and the subcellular context. We present discoveries of critical components required for Lm traversal of physiological barriers. Interactions between the host gut microbiota and Lm will be briefly discussed. Finally, the importance of Lm biodiversity and post-genomics approaches as a promising way to discover novel virulence factors will be highlighted.

Legionella pneumophila, the etiological agent of legionellosis, replicates within phagocytic cells. Crucial to biogenesis of the replicative vacuole is the Dot/Icm type 4 secretion system, which translocates a large number of effectors into the host cell cytosol. Among them is LegK2, a protein kinase that plays a key role in Legionella infection. Here, we identified the actin nucleator ARP2/3 complex as a target of LegK2. LegK2 phosphorylates the ARPC1B and ARP3 subunits of the ARP2/3 complex. LegK2-dependent ARP2/3 phosphorylation triggers global actin cytoskeleton remodeling in cells, and it impairs actin tail formation by Listeria monocytogenes, a well-known ARP2/3-dependent process. During infection, LegK2 is addressed to the Legionella-containing vacuole surface and inhibits actin polymerization on the phagosome, as revealed by legK2 gene inactivation. Consequently, LegK2 prevents late endosome/lysosome association with the phagosome and finally contributes to remodeling of the bacterium-containing phagosome into a replicative niche. The inhibition of actin polymerization by LegK2 and its effect on endosome trafficking are ARP2/3 dependent since it can be phenocopied by a specific chemical inhibitor of the ARP2/3 complex. Thus, LegK2-ARP2/3 interplay highlights an original mechanism of bacterial virulence with an unexpected role in local actin remodeling that allows bacteria to control vesicle trafficking in order to escape host defenses., IMPORTANCE Deciphering the individual contribution of each Dot/Icm type 4 secretion system substrate to the intracellular life-style of L. pneumophila remains the principal challenge in understanding the molecular basis of Legionella virulence. Our finding that LegK2 is a Dot/Icm effector that inhibits actin polymerization on the Legionella-containing vacuole importantly contributes to the deciphering of the molecular mechanisms evolved by Legionella to counteract the endocytic pathway. Indeed, our results highlight the essential role of LegK2 in preventing late endosomes from fusing with the phagosome. More generally, this work is the first demonstration of local actin remodeling as a mechanism used by bacteria to control organelle trafficking. Further, by characterizing the role of the bacterial protein kinase LegK2, we reinforce the concept that posttranslational modifications are key strategies used by pathogens to evade host cell defenses.

Efficient secretion of type III effector proteins from the bacterial cytoplasm to host cell cytosol via a type III secretion system (T3SS) is crucial for virulence of plant-pathogenic bacterium. Our previous study revealed a conserved hypothetical protein, virulence-related periplasm protein A (VrpA), which was identified as a critical virulence factor for Xanthomonas citri subsp. citri. In this study, we demonstrate that mutation of vrpA compromises X. citri subsp. citri virulence and hypersensitive response induction. This deficiency is also observed in the X. campestris pv. campestris strain, suggesting a functional conservation of VrpA in Xanthomonas spp. Our study indicates that VrpA is required for efficient protein secretion via T3SS, which is supported by multiple lines of evidence. A CyaA reporter assay shows that VrpA is involved in type III effector secretion; quantitative reverse-transcription polymerase chain reaction analysis suggests that the vrpA mutant fails to activate citrus-canker-susceptible gene CsLOB1, which is transcriptionally activated by transcription activator-like effector PthA4; in vitro secretion study reveals that VrpA plays an important role in secretion of T3SS pilus, translocon, and effector proteins. Our data also indicate that VrpA in X. citri subsp. citri localizes to bacterial periplasmic space and the periplasmic localization is required for full function of VrpA and X. citri subsp. citri virulence. Protein–protein interaction studies show that VrpA physically interacts with periplasmic T3SS components HrcJ and HrcC. However, the mutation of VrpA does not affect T3SS gene expression. Additionally, VrpA is involved in X. citri subsp. citri tolerance of oxidative stress. Our data contribute to the mechanical understanding of an important periplasmic protein VrpA in Xanthomonas spp.

Legionella pneumophila , an intracellular pathogen responsible for the severe pneumonia Legionnaires' disease, uses its dot/icm -encoded type IV secretion system (T4SS) to translocate effector proteins that promote its survival and replication into the host cell cytosol. However, by introducing bacterial products into the host cytosol, L. pneumophila also activates cytosolic immunosurveillance pathways, thereby triggering robust proinflammatory responses that mediate the control of infection. Thus, the pulmonary cell types that L. pneumophila infects not only may act as an intracellular niche that facilitates its pathogenesis but also may contribute to the immune response against L. pneumophila . The identity of these host cells remains poorly understood. Here, we developed a strain of L. pneumophila producing a fusion protein consisting of β-lactamase fused to the T4SS-translocated effector RalF, which allowed us to track cells injected by the T4SS. Our data reveal that alveolar macrophages and neutrophils both are the primary recipients of T4SS-translocated effectors and harbor viable L. pneumophila during pulmonary infection of mice. Moreover, both alveolar macrophages and neutrophils from infected mice produced tumor necrosis factor and interleukin-1α in response to T4SS-sufficient, but not T4SS-deficient, L. pneumophila . Collectively, our data suggest that alveolar macrophages and neutrophils are both an intracellular reservoir for L. pneumophila and a source of proinflammatory cytokines that contribute to the host immune response against L. pneumophila during pulmonary infection.

Microbial acquisition of nutrients in vivo is a fundamental aspect of infectious diseases, and is a potential target for anti-microbial therapy. Part of the innate host defense against microbial infection is nutritional restriction of access to sources of host nutrients (Abu Kwaik and Bumann, 2013; Eisenreich et al., 2013). Despite this host nutritional restriction, there has been a long held presumption that the host cell cytosol has sufficient nutrients for any intracellular pathogen, although many bacteria fail to grow in the host cytosol if they are microinjected (Goetz et al., 2001). However, recent studies on the two intra-vacuolar pathogens Anaplasma phagocytophilum (Niu et al., 2012) and Legionella pneumophila (Price et al., 2011) and the cytosolic pathogen Francisella tularensis (Steele et al., 2013) have clearly shown that the levels of amino acids in the host cell cytosol are below the threshold sufficient to meet the tremendous demands for carbon, nitrogen and energy to power the robust intracellular proliferation of these pathogens (Abu Kwaik and Bumann, 2013). Therefore, these intracellular pathogens have evolved with efficient strategies to boost the levels of host amino acids to meet their demands for higher levels of carbon, nitrogen and energy sources (Abu Kwaik and Bumann, 2013; Fonseca and Swanson, 2014). There is an emerging paradigm of specific microbial strategies that directly trigger the host cell to boost the cellular levels of essential microbial nutrients, and this paradigm has been designated as “nutritional virulence” (Abu Kwaik and Bumann, 2013). This opinion article is focused on nutritional virulence of L. pneumophila. In the aquatic environment, L. pneumophila proliferates within protozoa, which impact bacterial ecology and pathogenicity (Al-Quadan et al., 2012). Upon transmission to humans, L. pneumophila proliferates in alveolar macrophages within the Legionella-containing vacuole (LCV) that is ER-derived and evades lysosomal fusion (Figure ​(Figure1).1). Within both evolutionarily distant host cells, the Dot/Icm type IV secretion system of L. pneumophila injects ~300 protein effectors (Zhu et al., 2011; Luo, 2011a) that govern biogenesis of the LCV and modulate a myriad of cellular processes to enable intra-vacuolar proliferation (Figure ​(Figure1)1) (Luo, 2011b; Richards et al., 2013). Figure 1 Generation of a surplus of host amino acids by L. pneumophila and their import into the LCV. The AnkB effector is translocated into macrophages and amoebae by the Dot/Icm type IV secretion system of L. pneumophila. AnkB is immediately farnesylated and ... Amino acids are the main sources of carbon, nitrogen and energy for L. pneumophila, which metabolizes them through the TCA cycle (Pine et al., 1979), but also metabolizes minor amounts of glucose in vitro using the Entner-Doudoroff pathway (Eylert et al., 2010; Price et al., 2011). Although L. pneumophila utilizes amino acids as the main sources of carbon and energy, the pathogen is auxotrophic for seven amino acids (Cys, Met, Arg, Thr, Val, Ile, and Leu) (Eylert et al., 2010; Price et al., 2014). Remarkably, there is a high level of synchronization in amino acids auxotrophy between L. pneumophila and its host cells, which has likely played a factor in nutritional evolution of L. pneumophila as an intra-vacuolar pathogen (Price et al., 2014). Interestingly, intra-vacuolar L. pneumophila up-regulates its own amino acids transporters, indicating increased demands for amino acids in the intra-vacuolar environment (Bruggemann et al., 2006; Faucher et al., 2011; Eisenreich et al., 2013). Since the generation time of intra-vacuolar L. pneumophila is ~40 min, this organism requires high levels of amino acids to be imported from the host cytosol into the LCV lumen (Schunder et al., 2014). A long-held presumption has been that the host cell cytosol is rich in nutrients for invading pathogens. However, recent studies clearly indicate that the basal levels of host cellular amino acids are below the threshold sufficient for the robust intra-vacuolar proliferation of L. pneumophila (Sauer et al., 2005; Wieland et al., 2005) To achieve that needed threshold, L. pneumophila promotes host proteasomal degradation (Price et al., 2011) of LCV-decorated polyubiquitinated proteins (Dorer et al., 2006; Price et al., 2009, 2011; Lomma et al., 2010) mediated by the AnkB effector. Within human macrophages and amoeba, the AnkB translocated effector of L. pneumophila strain AA100/130b is localized to the cytosolic face of the LCV membrane through host-mediated farnesylation of its C-terminal CaaX motif (Figure ​(Figure1)1) (Price et al., 2010; Al-Quadan et al., 2011; Al-Quadan and Kwaik, 2011). On the LCV membrane, AnkB interacts with the host SCF1 ubiquitin ligase (Figure ​(Figure1)1) (Bruckert et al., 2014). As a bona fide F-box effector (Ensminger and Isberg, 2010; Lomma et al., 2010; Price and Abu Kwaik, 2010), AnkB triggers decoration of the LCV with Lys 48-linked polyubiquitinated proteins that are targeted for proteasomal degradation (Figure ​(Figure1)1) (Price et al., 2011). The metabolomic profile of L. pneumophila-infected amoeba and human cells have shown an AnkB-dependent dramatic rise in the levels of all cellular amino acids (Price et al., 2011), and this is initiated rapidly upon bacterial attachment to the macrophage plasma membrane (Bruckert et al., 2014). Importantly, inhibition of host proteasomal degradation abolishes intracellular proliferation of L. pneumophila strains AA100/130b and Philadelphia (Dorer et al., 2006; Price et al., 2011). The L. pneumophila-generated surplus of host cell amino acids may explain the lack of an intracellular defect for the lysine and tryptophan auxotrophic mutants of L. pneumophila (Mintz et al., 1988; Ensminger et al., 2012). Loss of AnkB in two independent isolates (AA100 and Paris) results in varying degrees of failure in intra-vacuolar proliferation and attenuation in the mouse model of Legionnaires' disease (Al-Khodor et al., 2008; Lomma et al., 2010). These defects for the AA100 strain are totally overcome upon supplementation of a mixture of amino acids in macrophages, amoeba and in the mouse model, similar to genetic complementation (Price et al., 2011). Importantly, silencing of the host SCF1 ubiquitin ligase, interference with Lys48-linked polyubiquitination, or inhibition of the host proteasomes block intra-vacuolar proliferation of L. pneumophila, but the block is relieved upon supplementation of an excess mixture of amino acids (Price et al., 2009, 2011). Surprisingly, the intra-vacuolar proliferation defect of the ankB mutant is rescued by many individual amino acids, such as Cys, Ala or Ser, which are essential or metabolically favorable for L. pneumophila (Pine et al., 1979). Interestingly, although Gln is the most abundant amino acid in human cells, supplementation of infected hMDMs with excess Gln alone efficiently rescues the ankB mutant (Price et al., 2011), while Glu is a major source of carbon and energy in vitro (Pine et al., 1979). These findings indicate that the basal levels of cellular amino acids are below the threshold sufficient for intra-vacuolar proliferation of L. pneumophila. Remarkably, pyruvate or citrate supplementation is as effective as amino acids in rescuing the intra-vacuolar growth defect of the ankB mutant, which indicates that the LCV is capable of importing these two substrates that can feed the TCA cycle, in addition to the documented reliance of intra-vacuolar L. pneumophila on amino acids (Schunder et al., 2014). In addition, L. pneumophila utilizes glucose through the Entner-Doudoroff pathway, which is required for proliferation within the amoeba host (Eylert et al., 2010). During inflammation, macrophages undergo up-regulation of glucose uptake and anaerobic glycolysis (Warburg-effect), which generates additional pyruvate (Eisenreich et al., 2013), and it is likely that both glucose and pyruvate are imported by the LCV. In addition, L. pneumophila-infected macrophages exhibit a pro-inflammatory phenotype. Taken together, it is likely that a multi-prong nutritional virulence strategy is utilized by L. pneumophila to generate and retrieve a diversified portfolio of sources of carbon and energy from the host cell. The host solute carrier (SLC) family of membrane proteins (Cedernaes et al., 2011; Schioth et al., 2013) that transport various compounds, including amino acids, TCA intermediates, glucose, lipids, and drugs are likely to be involved in import of various compounds by the LCV membrane (Figure ​(Figure1)1) (Wieland et al., 2005). Future studies should unravel the host metabolites and the mechanism of their import into the LCV lumen, and subsequently by the bacterial membrane. Deciphering microbial nutrition and metabolism in vivo is essential for our understanding of host-microbe interaction, and nutrient retrieval strategies by intracellular pathogens are potential targets for therapy.

A number of bacterial pathogens replicate within macrophages during host infections. Bacteria that establish a novel intracellular route either destroy the nascent phagosomal membrane to gain access to the host cell cytosol or else are targeted to a special membrane-bound compartment that allows exclusion of antimicrobial factors, and presumably access to nutrients. This latter group of pathogens includes Legionella pneumophila, Brucella abortus, Chlamydia species, and the eukaryotic parasite Toxoplasma gondii. The nascent phagosome formed by the coil has a distinct protein composition, an interesting observation likely to be important for subsequent events during intracellular growth. From a number of studies on the behavior of L. pneumophila within phagocytic cells, it is apparent that the phagosome bearing the microorganism is routed within the cell in a fashion distinct from that seen for phagosomes bearing nonpathogens. Protease mutants also appear to have virulences equal to those of wild-type microorganisms, although this result should be interpreted cautiously since partial differences in virulence or histopathology between the mutant and wild type may be difficult to detect in the guinea pig model. At this point, however, it can be said that the protease is dispensable for the intracellular trafficking pathway of L. pneumophila. Perhaps L. pneumophila, by disrupting the phagosomal membrane, causes a series of similar localized changes, with the result that the microorganism is eventually targeted to its replicative niche.

This chapter describes a series of interconnected problems for a range of bacterial, protozoal, and fungal pathogens and explores, in the order in which they are encountered by the pathogen, the consequences of each decision point in the establishment of an intracellular infection. There are three basic mechanisms of invasion: (i) phagocytosis, i.e., entry into professional phagocytes such as macrophages, monocytes, and neutrophils via a process dependent on the host cell contractile system; (ii) induced endocytosis and phagocytosis, i.e., entry into nonprofessional phagocytes by the active induction of internalization through the activity of the host cell contractile system; and (iii) active invasion, i.e., active entry into a passive host cell without triggering any contractile event in the host cell cytoskeleton. The niches exploited by intracellular pathogens fall readily into three different groupings. The first is intralysosomal, in which pathogens persist in acidic, hydrolytic compartments that interact with the endosomal network of the host. The second is intravacuolar, in which pathogens persist in nonacidic vacuoles that exhibit modified or little interaction with the endosomal system of the host. The third is cytoplasmic, in which pathogens exit the phagosome and reside within the host cell cytosol. This chapter has attempted to present the major points in the biology of these pathogens within a thematic framework from the time of initial infection, through the choice of intracellular niche, avoidance or exploitation of the immune response, and culminating in the metastasis or spread of the infection.

Caenorhabditis elegans is commonly used as an infection model for pathogenesis studies in Pseudomonas aeruginosa. The standard virulence assays rely on the slow and fast killing or paralysis of nematodes but here we developed a behaviour assay to monitor the preferred bacterial food sources of C. elegans. We monitored the food preferences of nematodes fed the wild type PAO1 and mutants in the type III secretion (T3S) system, which is a conserved mechanism to inject secreted effectors into the host cell cytosol. A ΔexsEΔpscD mutant defective for type III secretion served as a preferred food source, while an ΔexsE mutant that overexpresses the T3S effectors was avoided. Both food sources were ingested and observed in the gastrointestinal tract. Using the slow killing assay, we showed that the ΔexsEΔpscD had reduced virulence and thus confirmed that preferred food sources are less virulent than the wild type. Next we developed a high throughput feeding behaviour assay with 48 possible food colonies in order to screen a transposon mutant library and identify potential virulence genes. C. elegans identified and consumed preferred food colonies from a grid of 48 choices. The mutants identified as preferred food sources included known virulence genes, as well as novel genes not identified in previous C. elegans infection studies. Slow killing assays were performed and confirmed that several preferred food sources also showed reduced virulence. We propose that C. elegans feeding behaviour can be used as a sensitive indicator of virulence for P. aeruginosa PAO1.

Nucleotide-binding oligomerization domain protein (NOD)1 and NOD2 participate in signaling pathways that detect pathogen-induced processes, such as the presence of peptidoglycan fragments in the host cell cytosol, as danger signals. Recent work suggests that peptidoglycan fragments activate NOD1 indirectly, through activation of the small Rho GTPase Ras-related C3 botulinum toxin substrate 1 (RAC1). Excessive activation of small Rho GTPases by virulence factors of enteric pathogens also triggers the NOD1 signaling pathway. Many enteric pathogens use virulence factors that alter the activation state of small Rho GTPases, thereby manipulating the host cell cytoskeleton of intestinal epithelial cells to promote bacterial attachment or entry. These data suggest that the NOD1 signaling pathway in intestinal epithelial cells provides an important sentinel function for detecting 'breaking and entering' by enteric pathogens.

Background Australian bat lyssavirus (ABLV), a rhabdovirus of the genus Lyssavirus which circulates in both pteropid fruit bats and insectivorous bats in mainland Australia, has caused three fatal human infections, the most recent in February 2013, manifested as acute neurological disease indistinguishable from clinical rabies. Rhabdoviruses infect host cells through receptor-mediated endocytosis and subsequent pH-dependent fusion mediated by their single envelope glycoprotein (G), but the specific host factors and pathways involved in ABLV entry have not been determined. Methods ABLV internalization into HEK293T cells was examined using maxGFP-encoding recombinant vesicular stomatitis viruses (rVSV) that express ABLV G glycoproteins. A combination of chemical and molecular approaches was used to investigate the contribution of different endocytic pathways to ABLV entry. Dominant negative Rab GTPases were used to identify the endosomal compartment utilized by ABLV to gain entry into the host cell cytosol. Results Here we show that ABLV G-mediated entry into HEK293T cells was significantly inhibited by the dynamin-specific inhibitor dynasore, chlorpromazine, a drug that blocks clathrin-mediated endocytosis, and the actin depolymerizing drug latrunculin B. Over expression of dominant negative mutants of Eps15 and Rab5 also significantly reduced ABLV G-mediated entry into HEK293T cells. Chemical inhibitors of caveolae-dependent endocytosis and macropinocytosis and dominant negative mutants of Rab7 and Rab11 had no effect on ABLV entry. Conclusions The predominant pathway utilized by ABLV for internalization into HEK293T cells is clathrin-and actin-dependent. The requirement of Rab5 for productive infection indicates that ABLV G-mediated fusion occurs within the early endosome compartment.

The detection of bacterial pathogens by the innate immune system is mediated by various pattern recognition receptors that sense microbial molecules such as cell wall components, virulence factors or nucleic acids. Bacterial DNA is recognized by Toll-like receptor 9 (TLR9) at endosomal compartments and by cyclic GMP-AMP synthase, polymerase III/RIG-I, AIM2-like receptors (ALRs), DNA helicases as well as other incompletely characterized proteins in the host cell cytosol. Depending on the receptor and specific engagement of the adapter molecules MyD88, STING, MAVS or ASC, sensing of bacterial DNA triggers expression of NF-κB-dependent proinflammatory genes, type I IFN responses, and/or inflammasome activation. Whereas inflammatory gene expression and inflammasome activation are required for an effective host defense to most bacterial infections, type I IFNs appear to play a regulatory role and can be beneficial or detrimental for the host. This chapter summarizes the current knowledge about the mechanisms of DNA sensing and its function in bacterial infections.

Infection with Listeria monocytogenes strains that enter the host cell cytosol leads to a robust cytotoxic T cell response resulting in long-lived cell-mediated immunity (CMI). Upon entry into the cytosol, L. monocytogenes secretes cyclic diadenosine monophosphate (c-di-AMP) which activates the innate immune sensor STING leading to the expression of IFN-β and co-regulated genes. In this study, we examined the role of STING in the development of protective CMI to L. monocytogenes. Mice deficient for STING or its downstream effector IRF3 restricted a secondary lethal challenge with L. monocytogenes and exhibited enhanced immunity that was MyD88-independent. Conversely, enhancing STING activation during immunization by co-administration of c-di-AMP or by infection with a L. monocytogenes mutant that secretes elevated levels of c-di-AMP resulted in decreased protective immunity that was largely dependent on the type I interferon receptor. These data suggest that L. monocytogenes activation of STING downregulates CMI by induction of type I interferon., Author Summary Current vaccines are successful at generating neutralizing antibodies, however there is a pressing medical need to find adjuvants that yield long-lived memory T cells. Immunization with the bacterium Listeria monocytogenes induces a robust protective immune response mediated by cytotoxic lymphocytes that are efficient at killing infected cells upon reinfection. When L. monocytogenes enters a cell, it secretes the small molecule cyclic diadenosine monophosphate (c-di-AMP), which activates the host protein STING leading to a type I interferon response. In this study, we tested whether STING activation plays a role in the generation of cytotoxic lymphocytes and protective immunity using a mouse immunization model. We found that in the absence of STING signaling mice restricted bacterial growth and maintained higher numbers of cytotoxic lymphocytes upon reinfection, whereas mice immunized in the presence of elevated levels of c-di-AMP were less protected. These results suggest that the inflammation induced by a bacterial pathogen can be detrimental to the development of adaptive immunity, which could provide new insights into vaccine development.