Human genome-wide RNAi screen for host factors that modulate intracellular Salmonella growth

Salmonella enterica is a bacterial pathogen of humans that can proliferate within epithelial cells as well as professional phagocytes of the immune system. While much has been learned about the microbial genes that influence the infectious process through decades of intensive research, relatively little is known about the host factors that affect infection. We performed a genome-wide siRNA screen to identify host genes that Salmonella enterica serovar Typhimurium (S. typhimurium) utilizes to facilitate growth within human epithelial cells. In this screen, with siRNAs targeting every predicted gene in the human genome, we identified 252 new human-host-susceptibility factors (HSFs) for S. typhimurium. We also identified 39 genes whose silencing results in increased intracellular growth of S. typhimurium. The HSFs identified are regulated most centrally by NFκB and associate with each other through an extremely dense network of interactions that center around a group of kinases. Most genes identified were not previously appreciated as playing roles in the intracellular lifecycle of S. enterica. Numerous HSFs identified with interesting characteristics that could play plausible roles in mediating intracellular microbial growth are discussed. Importantly, this study reveals significant overlap between the host network that supports S. typhimurium growth within human epithelial cells and the one that promotes the growth of Mycobacterium tuberculosis within human macrophages. In addition to providing much new information about the molecular mechanisms underlying S. enterica-host cell interplay, all 252 HSFs identified are candidates for new anti-microbial targets for controlling S. enterica infections, and some may provide broad-spectrum anti-microbial activity.     

Manipulation of the host actin cytoskeleton by Salmonella--all in the name of entry

The invasive pathogen Salmonella enterica has evolved sophisticated mechanisms to subvert the cytoskeletal machinery of its host. Following contact with the host cell, it delivers a distinct arsenal of effector proteins directly into the cytoplasm. These bacterial effectors coordinate transient actin rearrangements and alter vesicle trafficking to trigger invasion, without causing overt cellular damage. Recent studies have shed new light on the signaling mechanisms underlying this remarkable host-pathogen interface, in particular, highlighting the unique multi-functional role and temporal regulation of key bacterial effectors.     

Biophysical characterization of SipA, an actin-binding protein from Salmonella enterica

An essential step in the pathogenesis of Salmonella enterica infections is bacterial entry into non-phagocytic cells of the intestinal epithelium. Proteins injected by Salmonella into host cells stimulate cellular responses that lead to extensive actin cytoskeleton reorganization and subsequent bacterial uptake. One of these proteins, SipA, modulates actin dynamics by directly binding to F-actin. We have biophysically characterized a C-terminal fragment, SipA(446-684), which has previously been shown to retain activity. Our results show that SipA(446-684) exhibits an elongated shape with a predominantly helical conformation and predict the existence of a coiled-coil domain. We suggest that the protein is able to span two adjacent actin monomers in a filament and propose a model that is consistent with the observed effects of SipA(446-684) on actin dynamics and F-actin stability and morphology.     

Persistent bacterial infections: the interface of the pathogen and the host immune system

Persistent bacterial infections involving Mycobacterium tuberculosis, Salmonella enterica serovar Typhi (S. typhi) and Helicobacter pylori pose significant public-health problems. Multidrug-resistant strains of M. tuberculosis and S. typhi are on the increase, and M. tuberculosis and S. typhi infections are often associated with HIV infection. This review discusses the strategies used by these bacteria during persistent infections that allow them to colonize specific sites in the host and evade immune surveillance. The nature of the host immune response to this type of infection and the balance between clearance of the pathogen and avoidance of damage to host tissues are also discussed.     

Chronic and acute infection of the gall bladder by Salmonella Typhi: understanding the carrier state

Despite major treatment and prevention efforts, millions of new typhoid infections occur worldwide each year. For a subset of infected individuals, Salmonella enterica subsp. enterica serovar Typhi colonizes the gall bladder and remains there long after symptoms subside, serving as a reservoir for the further spread of the disease. In this Progress article, we explore recent advances in our understanding of the mechanisms by which Salmonella spp.--predominantly S. Typhi--colonize and persist in the human gall bladder.     

Intracellular microbes and haemophagocytosis

Haemophagocytosis (hemophagocytosis) is the phenomenon of activated macrophage consumption of red and white blood cells, including professional phagocytes and lymphocytes. It can occur in patients with severe cases of intracellular microbial infection, including avian influenza, leishmaniasis, tuberculosis and typhoid fever. While well-known to physicians since at least the mid-1800s, haemophagocytosis has been little studied due to a paucity of tractable animal and cell culture models. Recently, haemophagocytosis has been described in a mouse model of typhoid fever, and it was noted that the infectious agent, Salmonella enterica, resides within haemophagocytic macrophages in mice. In addition, a cell culture model for haemophagocytosis revealed that S. enterica preferentially replicate in haemophagocytic macrophages. This review describes how, at the molecular and cellular levels, S. enterica may promote and take advantage of haemophagocytosis to establish long-term systemic infections in mammals. The role, relevance and possible molecular mechanisms of haemophagocytosis are discussed within the context of other microbial infections and of genetic deficiencies in which haemophagocytosis occurs and is associated with morbidity.     

Salmonella enterica serovar Typhimurium BipA exhibits two distinct ribosome binding modes

BipA is a highly conserved prokaryotic GTPase that functions to influence numerous cellular processes in bacteria. In Escherichia coli and Salmonella enterica serovar Typhimurium, BipA has been implicated in controlling bacterial motility, modulating attachment and effacement processes, and upregulating the expression of virulence genes and is also responsible for avoidance of host defense mechanisms. In addition, BipA is thought to be involved in bacterial stress responses, such as those associated with virulence, temperature, and symbiosis. Thus, BipA is necessary for securing bacterial survival and successful invasion of the host. Steady-state kinetic analysis and pelleting assays were used to assess the GTPase and ribosome-binding properties of S. enterica BipA. Under normal bacterial growth, BipA associates with the ribosome in the GTP-bound state. However, using sucrose density gradients, we demonstrate that the association of BipA and the ribosome is altered under stress conditions in bacteria similar to those experienced during virulence. The data show that this differential binding is brought about by the presence of ppGpp, an alarmone that signals the onset of stress-related events in bacteria.     

Role of antigens and virulence factors of Salmonella enterica serovar Typhi in its pathogenesis

Salmonella enterica serovar Typhi (S. Typhi), the aetiologic agent of typhoid fever, is a human restricted pathogen. The molecular mechanism of Salmonella pathogenicity is complex. The investigations of the molecular mechanisms of Salmonella virulence factors have shown that pathogenic Salmonella spp. are distinguished from their non-pathogenic relatives by the presence of specific pathogenicity genes, often organized in so-called pathogenicity islands (PIs). The type III secretion system (T3SS) proteins encoded by two Salmonella PIs (SPIs) are associated with the pathogenicity at molecular level. The identification of T3SS has provided new insight into the molecular factors and mechanisms underlying bacterial pathogenesis. The T3SS encoded by SPI-1 contains invasion genes; while SPI-2 is responsible for intracellular pathogenesis and has a crucial role for systemic S. enterica infections. These studies reveal a complex set of pathogenic interferences between intracellular Salmonella and its host cells. The understanding of the mechanisms by which Salmonella evade the host defense system and establish pathogenesis will be important for proper disease management.     

Manipulating cellular transport and immune responses: dynamic interactions between intracellular Salmonella enterica and its host cells

Intracellular survival and replication within eukaryotic host cells is of central importance for the pathogenesis of infections caused by Salmonella enterica. Intracellular Salmonella translocates a set of effector proteins by means of a type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2 (SPI2) that manipulates normal host-cell functions. Intracellular survival and replication is linked to the function of the SPI2-T3SS, but recent observations show that many additional cellular functions are targeted by this virulence system. In this review, we focus on the recent observations on the interference of intracellular Salmonella with functions of the innate and adaptive immune system and the modification of endocytic and exocytic cellular transport. The common molecular basis of the different SPI2-dependent phenotypes could be the interference with cellular transport along microtubules.     

Lack of O-antigen is essential for plasminogen activation by Yersinia pestis and Salmonella enterica

The O-antigen of lipopolysaccharide (LPS) is a virulence factor in enterobacterial infections, and the advantage of its genetic loss in the lethal pathogen Yersinia pestis has remained unresolved. Y. pestis and Salmonella enterica express beta-barrel surface proteases of the omptin family that activate human plasminogen. Plasminogen activation is central in pathogenesis of plague but has not, however, been found to be important in diarrhoeal disease. We observed that the presence of O-antigen repeats on wild-type or recombinant S. enterica, Yersinia pseudotuberculosis or Escherichia coli prevents plasminogen activation by PgtE of S. enterica and Pla of Y. pestis; the O-antigen did not affect incorporation of the omptins into the bacterial outer membrane. Purified His6-Pla was successfully reconstituted with rough LPS but remained inactive after reconstitution with smooth LPS. Expression of smooth LPS prevented Pla-mediated adhesion of recombinant E. coli to basement membrane as well as invasion into human endothelial cells. Similarly, the presence of an O-antigen prevented PgtE-mediated bacterial adhesion to basement membrane. Substitution of Arg-138 and Arg-171 of the motif for protein binding to lipid A 4'-phosphate abolished proteolytic activity but not membrane translocation of PgtE, indicating dependence of omptin activity on a specific interaction with lipid A. The results suggest that Pla and PgtE require LPS for activity and that the O-antigen sterically prevents recognition of large-molecular-weight substrates. Loss of O-antigen facilitates Pla functions and invasiveness of Y. pestis; on the other hand, smooth LPS renders plasminogen activator cryptic in S. enterica.     

Insect cellular reactions to the lipopolysaccharide component of the bacterium Serratia marcescens are mediated by eicosanoids

Nodulation, which begins with the formation of cellular microaggregates, is the predominant cellular defense reaction to bacterial infections in insects. We suggested that these reactions to bacterial infections are mediated by eicosanoids. The lipopolysaccharide (LPS) component of some bacterial cells stimulates defense reactions in mammals and insects. Here, we report on experiments designed to test the hypothesis that eicosanoids mediate microaggregation reactions to LPS. Injections of LPS (purified from the bacterium, Serratia marcescens) into larvae of the tenebrionid beetle, Zophobas atratus, stimulated microaggregation reactions in a dose-dependent manner. Treatments with eicosanoid-biosynthesis inhibitors immediately prior to LPS challenge sharply reduced the microaggregation responses. Separate treatments with specific inhibitors of phospholipase A(2), cyclooxygenase and lipoxygenase reduced microaggregation, supporting our view that microaggregate formation involves lipoxygenase and cyclooxygenase products. The inhibitory influence of dexamethasone was apparent within 30min after injection, and microaggregation was significantly reduced, relative to control insects, over the following 90min. The dexamethasone effects were reversed by treating LPS-injected insects with the eicosanoid precursor, arachidonic acid. These findings indicate that cellular defense reactions to a specific component of bacterial cells are mediated by eicosanoids, and open up new possibilities for dissecting detailed hemocytic actions in insect immune reactions to bacterial infections.     

Crystal structure of a novel prokaryotic Ser/Thr kinase and its implication in the Cpx stress response pathway

The Cpx signalling system of Escherichia coli and Salmonella enterica senses extracytoplasmic stress and controls expression of factors that allow the bacterium to adapt to these stressors and thereby enhance survival. Many of the Cpx-responsive genes products are of unknown function. We determined the crystal structure of one of these gene products, called YihE in E. coli, which exhibits a eukaryotic kinase fold. Functional assays established that both YihE and the S. enterica YihE homologue, RdoA, undergo autophosphorylation and phosphorylate protein substrates at Ser/Thr residues in vitro, demonstrating that YihE/RdoA is a novel Ser/Thr protein kinase in prokaryotic cells. Phenotypic analysis of yihE/rdoA null strains indicates that this kinase is most abundant in stationary phase, and is important for long-term cell survival and for expression of surface appendages in both a Cpx-independent and -dependent manner. YihE/RdoA is therefore a previously unknown kinase component of a new type of bacterial phosphorelay mechanism, adding kinase activity as another response to the Cpx sensing system that functions to maintain cellular homeostasis.     

Salmonella type III secretion-associated protein InvE controls translocation of effector proteins into host cells

Salmonella enterica encodes a type III secretion system (TTSS) within a pathogenicity island located at centisome 63 (SPI-1), which is essential for its pathogenicity. This system mediates the transfer of a battery of bacterial proteins into the host cell with the capacity to modulate cellular functions. The transfer process is dependent on the function of protein translocases SipB, SipC, and SipD. We report here that Salmonella protein InvE, which is also encoded within SPI-1, is essential for the translocation of bacterial proteins into host cells. An S. enterica serovar Typhimurium mutant carrying a loss-of-function mutation in invE shows reduced secretion of SipB, SipC, and SipD while exhibiting increased secretion of other TTSS effector proteins. We also demonstrate that InvE interacts with a protein complex formed by SipB, SipC, and their cognate chaperone, SicA. We propose that InvE controls protein translocation by regulating the function of the Sip protein translocases.     

Hemophagocytic macrophages harbor Salmonella enterica during persistent infection

Salmonella enterica subspecies can establish persistent, systemic infections in mammals, including human typhoid fever. Persistent S. enterica disease is characterized by an initial acute infection that develops into an asymptomatic chronic infection. During both the acute and persistent stages, the bacteria generally reside within professional phagocytes, usually macrophages. It is unclear how salmonellae can survive within macrophages, cells that evolved, in part, to destroy pathogens. Evidence is presented that during the establishment of persistent murine infection, macrophages that contain S. enterica serotype Typhimurium are hemophagocytic. Hemophagocytic macrophages are characterized by the ingestion of non-apoptotic cells of the hematopoietic lineage and are a clinical marker of typhoid fever as well as certain other infectious and genetic diseases. Cell culture assays were developed to evaluate bacterial survival in hemophagocytic macrophages. S. Typhimurium preferentially replicated in macrophages that pre-phagocytosed viable cells, but the bacteria were killed in macrophages that pre-phagocytosed beads or dead cells. These data suggest that during persistent infection hemophagocytic macrophages may provide S. Typhimurium with a survival niche.     

Rho GTPase-activating bacterial toxins: from bacterial virulence regulation to eukaryotic cell biology

Studies on the interactions of bacterial pathogens with their host have provided an invaluable source of information on the major functions of eukaryotic and prokaryotic cell biology. In addition, this expanding field of research, known as cellular microbiology, has revealed fascinating examples of trans-kingdom functional interplay. Bacterial factors actually exploit eukaryotic cell machineries using refined molecular strategies to promote invasion and proliferation within their host. Here, we review a family of bacterial toxins that modulate their activity in eukaryotic cells by activating Rho GTPases and exploiting the ubiquitin/proteasome machineries. This family, found in human and animal pathogenic Gram-negative bacteria, encompasses the cytotoxic necrotizing factors (CNFs) from Escherichia coli and Yersinia species as well as dermonecrotic toxins from Bordetella species. We survey the genetics, biochemistry, molecular and cellular biology of these bacterial factors from the standpoint of the CNF1 toxin, the paradigm of Rho GTPase-activating toxins produced by urinary tract infections causing pathogenic Escherichia coli. Because it reveals important connections between bacterial invasion and the host inflammatory response, the mode of action of CNF1 and its related Rho GTPase-targetting toxins addresses major issues of basic and medical research and constitutes a privileged experimental model for host-pathogen interaction.     

How do bacteria resist human antimicrobial peptides?

Cationic antimicrobial peptides (CAMPs), such as defensins, cathelicidins and thrombocidins, are an important human defense mechanism, protecting skin and epithelia against invading microorganisms and assisting neutrophils and platelets. Staphylococcus aureus, Salmonella enterica and other bacterial pathogens have evolved countermeasures to limit the effectiveness of CAMPs, including the repulsion of CAMPs by reducing the net negative charge of the bacterial cell envelope through covalent modification of anionic molecules (e.g. teichoic acids, phospholipids and lipid A); expelling CAMPs through energy-dependent pumps; altering membrane fluidity; and cleaving CAMPs with proteases. Mutants susceptible to CAMPs are more efficiently inactivated by phagocytes and are virulence-attenuated, indicating that CAMP resistance plays a key role in bacterial infections.     

Colonization of tomato plants by Salmonella enterica is cultivar dependent, and type 1 trichomes are preferred colonization sites

Nontyphoid salmonellosis caused by Salmonella enterica is the most common bacterial food-borne illness in humans, and fresh produce, including tomatoes, is a common vehicle. Accumulating data indicate that human enteric pathogenic bacteria, including S. enterica, interact actively with plants. Tomato plants were inoculated with S. enterica to evaluate plausible contamination routes and to determine if the tomato cultivar affects S. enterica colonization. S. enterica population levels on tomato leaves were cultivar dependent. S. enterica levels on Solanum pimpinellifolium (West Virginia 700 [WVa700]) were lower than on S. lycopersicum cultivars. S. enterica preferentially colonized type 1 trichomes and rarely interacted with stomata, unlike what has been reported for cut lettuce leaves. Early S. enterica leaf colonization led to contamination of all fruit, with levels as high as 10(5) CFU per fruit. Reduced bacterial speck lesion formation correlated with reduced S. enterica populations in the phyllosphere. Tomato pedicels and calyxes also harbored large S. enterica populations following inoculation via contaminated water postharvest. WVa700 green fruit harbored significantly smaller S. enterica populations than did red fruit or S. lycopersicum fruit. We found that plants irrigated with contaminated water had larger S. enterica populations than plants grown from seeds planted in infested soil. However, both routes of contamination resulted in detectable S. enterica populations in the phyllosphere. Phyllosphere S. enterica populations pose a risk of fruit contamination and subsequent human disease. Restricting S. enterica phyllosphere populations may result in reduced fruit contamination. We have identified WVa700 as a tomato cultivar that can restrict S. enterica survival in the phyllosphere.     

The evolving genome of Salmonella enterica serovar Pullorum

Salmonella enterica serovar Pullorum is a fowl-adapted bacterial pathogen that causes dysentery (pullorum disease). Host adaptation and special pathogenesis make S. enterica serovar Pullorum an exceptionally good system for studies of bacterial evolution and speciation, especially regarding pathogen-host interactions and the acquisition of pathogenicity. We constructed a genome map of S. enterica serovar Pullorum RKS5078, using I-CeuI, XbaI, AvrII, and SpeI and Tn10 insertions. Pulsed-field gel electrophoresis was employed to separate the large DNA fragments generated by the endonucleases. The genome is 4,930 kb, which is similar to most salmonellas. However, the genome of S. enterica serovar Pullorum RKS5078 is organized very differently from the majority of salmonellas, with three major inversions and one translocation. This extraordinary genome structure was seen in most S. enterica serovar Pullorum strains examined, with different structures in a minority of S. enterica serovar Pullorum strains. We describe the coexistence of different genome structures among the same bacteria as genomic plasticity. Through comparisons with S. enterica serovar Typhimurium, we resolved seven putative insertions and eight deletions ranging in size from 12 to 157 kb. The genomic plasticity seen among S. enterica serovar Pullorum strains supported our hypothesis about its association with bacterial evolution: a large genomic insertion (157 kb in this case) disrupted the genomic balance, and rebalancing by independent recombination events in individual lineages resulted in diverse genome structures. As far as the structural plasticity exists, the S. enterica serovar Pullorum genome will continue evolving to reach a further streamlined and balanced structure.     

感染在神经退行性疾病中的调控机制

神经退行性疾病以突触丢失和神经元死亡为特征,表现为认知功能下降、痴呆和运动功能丧失.流行病学和实验证据提示:慢性细菌、病毒和真菌感染可能是导致神经退行性疾病如阿尔兹海默症(AD)、帕金森病(PD)、肌萎缩性侧索硬化症(ALS)和多发性硬化症(MS)等的危险因素.病原体在中枢神经系统的持续感染可导致一系列细胞生物学功能的...     

Influence of human gastrointestinal tract bacterial pathogens on host cell apoptosis

Several pathogenic bacteria are able to trigger apoptosis in the host cell, but the mechanisms by which it occurs differ, and the resulting pathology can take different courses. Induction and/or blockage of programmed cell death upon infection is a result of complex interaction of bacterial proteins with cellular proteins involved in signal transduction and apoptosis. In this review we focus on pro/anti-apoptotic activities exhibited by two enteric pathogens Salmonella enterica, Yersinia spp. and gastric pathogen Helicobacter pylori. We present current knowledge on how interaction between mammalian and bacterial cell relates to the molecular pathways of apoptosis, and what is the role of apoptosis in pathogenesis.