Salmonella Typhimurium has evolved a complex functional interface with its host cell largely determined by two type III secretion systems (T3SS), which through the delivery of bacterial effector proteins modulate a variety of cellular processes. We show here that Salmonella Typhimurium infection of epithelial cells results in a profound transcriptional reprogramming that changes over time. This response is triggered by Salmonella T3SS effector proteins, which stimulate unique signal transduction pathways leading to STAT3 activation. We found that the Salmonella-stimulated changes in host cell gene expression are required for the formation of its specialized vesicular compartment that is permissive for its intracellular replication. This study uncovers a cell-autonomous process required for Salmonella pathogenesis potentially opening up new avenues for the development of anti-infective strategies that target relevant host pathways.
Essential for the ability of Salmonella Typhimurium to cause disease is the function of a type III secretion system (T3SS) encoded within its pathogenicity island 1 (SPI-1), which through the delivery of bacterial effector proteins modulates a variety of cellular functions. This study reports that the infection of mammalian cells with Salmonella Typhimurium results in a profound reprogramming of gene expression that changes over time. The stimulation of this response requires the activity of a specific subset of bacterial T3SS effector proteins, which stimulate unique signal transduction pathways leading to STAT3 activation. We found that the Salmonella-stimulated changes in host cell gene expression are required for its intracellular replication. Targeting the mechanisms described in this study may lead to the development of novel anti-infective strategies.
Type III protein secretion systems are being considered for vaccine development since virtually any protein antigen can be engineered for delivery by these nanomachines into the class I antigen presentation pathway to stimulate antigen-specific CD8+ T cells 12. A limitation in the use of this system is that it requires live virulence-attenuated bacteria, which may preclude its use in certain populations such as children and the immunocompromised. Here we report the engineering of the Salmonella Typhimurium type III secretion system in achromosomal, non-replicating nanoparticles derived from bacterial minicells. The engineered system is shown to be functional and capable of delivering heterologous antigens to the class I antigen presentation pathway stimulating immune responses both in vitro and in vivo. This antigen delivery platform offers a novel approach for vaccine development and cellular immunotherapy.
Unlike other Salmonellae, the intracellular bacterial human pathogen Salmonella Typhi exhibits strict host specificity. The molecular bases for this restriction are unknown. Here we show that the expression of a single type III secretion system effector protein from broad-host Salmonella Typhimurium allows Salmonella Typhi to survive and replicate within macrophages and tissues from mice, a non-permissive host. We found that this effector proteolytically targets Rab32, which controls traffic to lysosome-related organelles in conjunction with components of the biogenesis of lysosome-related organelle complexes (BLOCs). RNAi-mediated depletion of Rab32 or of an essential component of a BLOC complex was sufficient to allow S. Typhi to survive within mouse macrophages. Furthermore, Salmonella Typhi was able to survive in macrophages from mice defective in BLOC components. These findings provide insight into the molecular bases of S. Typhi host restriction and uncover a previously unknown mechanism of pathogen control in macrophages.
typhoid fever; Rab GTPases; Rab32; membrane traffic; bacterial pathogenesis; type III secretion; macrophages; innate immunity; lysosomes; lysosome-related organelles
Ubiquitinylation of proteins is a critical mechanism in regulating numerous eukaryotic cellular processes including cell cycle progression, inflammatory response, and vesicular trafficking. Given the importance of ubiquitinylation, it is not surprising that several pathogenic bacteria have developed strategies to exploit various stages of the ubiquitin pathway for their own benefit. One such strategy is the delivery of bacterial ‘effector’ proteins into the host cell cytosol, which mimic the activities of components of the host ubiquitin pathway. Recent studies have highlighted a number of bacterial effectors that functionally mimic the activity of eukaryotic E3 ubiquitin ligases, including a novel structural class of bacterial E3 ligases that provides a striking example of convergent evolution.
Salmonella enterica, the cause of food poisoning and typhoid fever, has evolved sophisticated mechanisms to modulate Rho family guanosine triphosphatases (GTPases) to mediate specific cellular responses such as actin remodeling, macropinocytosis, and nuclear responses. These responses are largely the result of the activity of a set of bacterial proteins (SopE, SopE2, and SopB) that, upon delivery into host cells via a type III secretion system, activate specific Rho family GTPases either directly (SopE and SopE2) or indirectly (SopB) through the stimulation of an endogenous exchange factor. We show that different Rho family GTPases play a distinct role in Salmonella-induced cellular responses. In addition, we report that SopB stimulates cellular responses by activating SH3-containing guanine nucleotide exchange factor (SGEF), an exchange factor for RhoG, which we found plays a central role in the actin cytoskeleton remodeling stimulated by Salmonella. These results reveal a remarkable level of complexity in the manipulation of Rho family GTPases by a bacterial pathogen.
Caspase-1 is activated by a variety of stimuli after the assembly of the “inflammasome,” an activating platform made up of a complex of the NOD-LRR family of proteins. Caspase-1 is required for the secretion of proinflammatory cytokines, such as interleukin (IL)-1β and IL-18, and is involved in the control of many bacterial infections. Paradoxically, however, its absence has been reported to confer resistance to oral infection by Salmonella typhimurium. We show here that absence of caspase-1 or components of the inflammasome does not result in resistance to oral infection by S. typhimurium, but rather, leads to increased susceptibility to infection.
Salmonella typhimurium has sustained a long-standing association with its host and therefore has evolved sophisticated strategies to multiply and survive within this environment. Central to Salmonella pathogenesis is the function of a dedicated type III secretion system that delivers bacterial effector proteins into the host cell cytoplasm. These effectors stimulate nuclear responses and actin cytoskeleton reorganization leading to the production of proinflammatory cytokines and bacterial internalization. The stimulation of these responses requires the function of Cdc42, a member of the Rho family of small molecular weight GTPases, and SopE, a bacterial effector protein that stimulates guanine nucleotide exchange on Rho GTPases. However, nothing is known about the role of Cdc42 effector proteins in S. typhimurium–induced responses. We showed here that S. typhimurium infection of cultured epithelial cells results in the activation of p21-activated kinase (PAK), a serine/threonine kinase that is an effector of Cdc42-dependent responses. Transient expression of a kinase-defective PAK blocked both S. typhimurium– and SopE-induced c-Jun NH2-terminal kinase (JNK) activation but did not interfere with bacteria-induced actin cytoskeleton rearrangements. Similarly, expression of SH3-binding mutants of PAK did not block actin-mediated S. typhimurium entry into cultured cells. However, expression of an effector loop mutant of Cdc42Hs (Cdc42HsC40) unable to bind PAK and other CRIB (for Cdc42/Rac interacting binding)-containing target proteins resulted in abrogation of both S. typhimurium–induced nuclear and cytoskeletal responses. These results show that PAK kinase activity is required for bacteria-induced nuclear responses but it is not required for cytoskeletal rearrangements, indicating that S. typhimurium stimulates cellular responses through different Cdc42 downstream effector activities. In addition, these results demonstrate that the effector loop of Cdc42 implicated in the binding of PAK and other CRIB-containing target proteins is required for both responses.
Cdc42; signal transduction; actin cytoskeleton; bacterial pathogenesis
Campylobacter jejuni is the major cause of bacterial food-borne illness in the USA and Europe. An important virulence attribute of this bacterial pathogen is its ability to enter and survive within host cells. Here we show through a quantitative proteomic analysis that upon entry into host cells, C. jejuni undergoes a significant metabolic downshift. Furthermore, our results indicate that intracellular C. jejuni reprograms its respiration, favoring the respiration of fumarate. These results explain the poor ability of C. jejuni obtained from infected cells to grow under standard laboratory conditions and provide the bases for the development of novel anti microbial strategies that would target relevant metabolic pathways.
Campylobacter jejuni is one of the most common causes of food-borne illness in the United States and a major cause of diarrheal diseases in developing countries. This pathogen can invade intestinal epithelial cells, which is very important for its ability to cause disease. Once it gains access to epithelial cells, C. jejuni becomes unable to grow under standard growth conditions, although it can grow if pre-incubated under oxygen limiting conditions. This study compares the protein compositions of C. jejuni grown under standard growth conditions and obtained from within epithelial cells. This analysis indicates that, within cells, C. jejuni undergoes a significant metabolic downshift and reprograms its respiration, favoring the respiration of fumarate. These results may provide the bases for the development of novel anti microbial strategies that would target relevant metabolic pathways.
The intracellular pathogen Legionella pneumophila modulates the activity of host GTPases to direct the transport and assembly of the membrane-bound compartment in which it resides1–6. In vitro studies have suggested that the Legionella protein DrrA post-translationally modifies the GTPase Rab1 by a process called AMPylation7. Here, mass spectrometry was used to investigate post-translational modifications to Rab1 that occur during infection of host cells by Legionella. Consistent with in vitro studies, DrrA-mediated AMPylation of a conserved tyrosine residue in the switch II region of Rab1 was detected during infection. In addition, a modification to an adjacent serine residue in Rab1 was discovered, which was independent of DrrA. The Legionella effector protein AnkX was required for this modification. Biochemical studies determined that AnkX directly mediates the covalent attachment of a phosphocholine moiety to Rab1. This phosphocholine transferase activity used CDP-choline as a substrate and required a conserved histidine residue located in the FIC domain of the AnkX protein. During infection, AnkX modified both Rab1 and Rab35, which explains how this protein modulates membrane transport through both the endocytic and exocytic pathways of the host cell. Thus, phosphocholination of Rab GTPases represents a mechanism by which bacterial FIC domain-containing proteins can alter host cell functions.
Salmonella enterica serovar Typhi (S. Typhi) is the cause of typhoid fever, a life-threatening disease of humans. The lack of an animal model due to S. typhi's strict human host specificity has been a significant obstacle in the understanding of its pathogenesis and the development of a safe and effective vaccine against typhoid fever. We report here the development of a mouse model for S. Typhi infection. We showed that immunodeficient Rag2 -/- γc -/- mice engrafted with human fetal liver hematopoietic stem and progenitor cells were able to support S. Typhi replication and persistent infection. A S. Typhi strain carrying a mutation in a gene required for its virulence in humans was not able to replicate in these humanized mice. In contrast, another mutant strain unable to produce the recently identified typhoid toxin, exhibited increased replication suggesting a potential role for this toxin in the establishment of persistent infection. Furthermore, infected animals mounted a human innate and adaptive immune response to S. Typhi resulting in the production of cytokines and pathogen-specific antibodies. These results therefore indicate that this animal model can be used to study S. Typhi pathogenesis and to evaluate potential vaccine candidates against typhoid fever.
bacterial pathogenesis; innate immunity; stem cells; humanized mouse; typhoid fever
Campylobacter jejuni is the leading cause of infectious gastroenteritis in industrialized nations. Its ability to enter and survive within nonphagocytic cells is thought to be very important for pathogenesis. However, little is known about the C. jejuni determinants that mediate these processes. Through an extensive transposon mutagenesis screen, we have identified several loci that are required for C. jejuni efficient entry and survival within epithelial cells. Among these loci, insertional mutations in aspA, aspB, and sodB resulted in drastic reduction in C. jejuni entry and/or survival within host cells and a severe defect in colonization in an animal model. The implications of these findings for the understanding of C. jejuni-host cell interactions are discussed.
Central to the biology of many pathogenic bacteria are a number of specialized machines, known as type III, type IV or type VI protein secretion systems. These machines have specifically evolved to deliver bacterial “effector” proteins into host cells with the capacity to modulate a variety of cellular functions. The identification of the biochemical activities of many effector proteins, coupled with a better understanding of their potential contribution to pathogenesis, have revealed common themes in the evolutionary design and function of these remarkable bacterial proteins.
bacterial pathogenesis; Type III, Type IV and Type VI secretion systems; pathogen evolution; host-pathogen interactions
Campylobacter jejuni is a leading cause of food-borne illness in the United States. Despite significant recent advances, its mechanisms of pathogenesis are poorly understood. A unique feature of this pathogen is that, with some exceptions, it lacks homologs of known virulence factors from other pathogens. Through a genetic screen, we have identified a C. jejuni homolog of the VirK family of virulence factors, which is essential for antimicrobial peptide resistance and mouse virulence.
Many bacterial pathogens and symbionts utilize type III secretion systems to deliver bacterial effector proteins into host cells. These effector proteins have the capacity to modulate a large variety of cellular functions in a highly regulated manner. Here we report that the phosphoinositide phosphatase SopB, a Salmonella Typhimurium type III secreted effector protein, diversifies its function by localizing to different cellular compartments in a ubiquitin-dependent manner. We show that SopB utilizes the same enzymatic activity to modulate actin-mediated bacterial internalization and Akt activation at the plasma membrane, and vesicular traffic and intracellular bacterial replication at the phagosome. Thus by exploiting the host cellular machinery, Salmonella Typhimurium has evolved the capacity to broaden the functional repertoire of a virulence factor to maximize its ability to modulate cellular functions.
bacterial pathogenesis; phosphoinositide phosphatase; type III secretion; vesicular trafficking; actin remodeling
The correct organization of single subunits of multi-protein machines in a three dimensional context is critical for their functionality. Type III secretion systems (T3SS) are molecular machines with the capacity to deliver bacterial effector proteins into host cells and are fundamental for the biology of many pathogenic or symbiotic bacteria. A central component of T3SSs is the needle complex, a multiprotein structure that mediates the passage of effector proteins through the bacterial envelope. We have used cryo electron microscopy combined with bacterial genetics, site-specific labeling, mutational analysis, chemical derivatization and high-resolution mass spectrometry to generate an experimentally validated topographic map of a Salmonella typhimurium T3SS needle complex. This study provides insights into the organization of this evolutionary highly conserved nanomachinery and is the basis for further functional analysis.
Many Gram negative pathogens such as Salmonella, Yersinia, or Shigella use the type III secretion system (T3SS) to initiate infection in eukaryotic cells, resulting in well known clinical symptoms ranging from mild headaches and diarrhea to life-threatening diseases such as typhoid fever or bubonic plague. The T3SS is a highly developed macromolecular system that serves as a platform to make physical contact between host cells and pathogens and mediates the translocation of bacterial toxins (effector proteins) into eukaryotic cells. Central to the T3SS is the mega-dalton sized membrane associated needle complex, which is composed of several soluble and membrane proteins; however, their organization within the needle complex critical for proper assembly and function is unclear. Here, we use an integrated experimental approach that combines cryo electron microscopy with bacterial genetics, site-specific labeling, mutational analysis, chemical derivatization and high-resolution mass spectrometry in order to determine the topographic organization of individual components of the Salmonella typhimurium needle complex and define sites critical for its stability. Our study provides insights into the organization of this evolutionary highly conserved system and is the basis for further functional analysis.
Delivery of bacterial proteins into mammalian cells by type III secretion systems (TTSS) is thought to require the intimate association of bacteria with target cells. The molecular bases of this intimate association appear to be different in different bacteria involving TTSS components, as well as surface determinants not associated with TTSS. We show here that the protein translocases SipB, SipC, and SipD of the Salmonella enterica serovar Typhimurium pathogenicity island 1 (SPI-1)-encoded TTSS are required for the intimate association of these bacteria with mammalian cells. S. Typhimurium mutant strains lacking any of the translocases were defective for intimate attachment. Immunofluorescence microscopy showed that SipD is present on the bacterial surface prior to bacterial contact with host cells. In contrast, SipB and SipC were detected on the bacterial surface only subsequent to bacterial contact with the target cell. We conclude that the coordinated deployment and interaction between the protein translocases mediate the SPI-1 TTSS-dependent intimate association of S. Typhimurium with host cells.
Salmonella enterica utilizes a type III secretion system (TTSS) encoded in its pathogenicity island 1 to mediate its initial interactions with intestinal epithelial cells, which are characterized by the stimulation of actin cytoskeleton reorganization and a profound reprogramming of gene expression. These responses result from the stimulation of Rho-family GTPases and downstream signaling pathways by specific effector proteins delivered by this TTSS. We show here that AvrA, an effector protein of this TTSS, specifically inhibits the Salmonella-induced activation of the JNK pathway through its interaction with MKK7, although it does not interfere with the bacterial infection-induced NF-κB activation. We also show that AvrA is phosphorylated at evolutionary conserved residues by a TTSS-effector-activated ERK pathway. This interplay between effector proteins delivered by the same TTSS highlights the remarkable complexity of these systems.
Salmonella Typhimurium is a major cause of diarrheal disease worldwide. Central to S. Typhimurium's pathogenesis is its ability to induce intestinal inflammation, which is initiated by several bacterial proteins injected into intestinal epithelial cells by a nanomachine known as the type III secretion system. We show here that another bacterial protein injected by this machine negatively influences the responses triggered by Salmonella, presumably to limit cellular damage. This interplay between bacterial proteins of opposite function highlights the remarkable complexity of the host/pathogen interface.
Recognition of conserved bacterial products by innate immune receptors leads to inflammatory responses that control pathogen spread but that can also result in pathology. Intestinal epithelial cells are exposed to bacterial products and therefore must prevent signaling through innate immune receptors to avoid pathology. However, enteric pathogens are able to stimulate intestinal inflammation. We show here that the enteric pathogen Salmonella Typhimurium can stimulate innate immune responses in cultured epithelial cells by mechanisms that do not involve receptors of the innate immune system. Instead, S. Typhimurium stimulates these responses by delivering through its type III secretion system the bacterial effector proteins SopE, SopE2, and SopB, which in a redundant fashion stimulate Rho-family GTPases leading to the activation of mitogen-activated protein (MAP) kinase and NF-κB signaling. These observations have implications for the understanding of the mechanisms by which Salmonella Typhimurium induces intestinal inflammation as well as other intestinal inflammatory pathologies.
Salmonella Typhimurium is one of the most common causes of food-borne illness in the United States and a major cause of diarrheal diseases in developing countries. This pathogen induces diarrhea by stimulating inflammation in the intestinal tract. This study shows that S. Typhimurium delivers molecules into epithelial cells with the capacity to stimulate the production of pro-inflammatory substances. This mechanism may help the pathogen to initiate the inflammatory response in the intestinal epithelium. This study provides insight into the mechanisms by which Salmonella Typhimurium causes diarrhea.
Bacterial type III secretion machines have adapted to carry out numerous functions, ranging from locomotion to protein delivery into nucleated cells. One of the most intriguing issues is the source of energy that fuels their activities. Despite recent advances, there are still many questions to be resolved.
Microbial pathogenesis studies traditionally encompass dissection of virulence properties such as the bacterium's ability to elaborate toxins, adhere to and invade host cells, cause tissue damage, or otherwise disrupt normal host immune and cellular functions. In contrast, bacterial metabolism during infection has only been recently appreciated to contribute to persistence as much as their virulence properties. In this study, we used comparative proteomics to investigate the expression of uropathogenic Escherichia coli (UPEC) cytoplasmic proteins during growth in the urinary tract environment and systematic disruption of central metabolic pathways to better understand bacterial metabolism during infection. Using two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) and tandem mass spectrometry, it was found that UPEC differentially expresses 84 cytoplasmic proteins between growth in LB medium and growth in human urine (P<0.005). Proteins induced during growth in urine included those involved in the import of short peptides and enzymes required for the transport and catabolism of sialic acid, gluconate, and the pentose sugars xylose and arabinose. Proteins required for the biosynthesis of arginine and serine along with the enzyme agmatinase that is used to produce the polyamine putrescine were also up-regulated in urine. To complement these data, we constructed mutants in these genes and created mutants defective in each central metabolic pathway and tested the relative fitness of these UPEC mutants in vivo in an infection model. Import of peptides, gluconeogenesis, and the tricarboxylic acid cycle are required for E. coli fitness during urinary tract infection while glycolysis, both the non-oxidative and oxidative branches of the pentose phosphate pathway, and the Entner-Doudoroff pathway were dispensable in vivo. These findings suggest that peptides and amino acids are the primary carbon source for E. coli during infection of the urinary tract. Because anaplerosis, or using central pathways to replenish metabolic intermediates, is required for UPEC fitness in vivo, we propose that central metabolic pathways of bacteria could be considered critical components of virulence for pathogenic microbes.
Bacteria that cause infections often have genes known as virulence factors that are required for bacteria to cause disease. Studying virulence factors such as toxins, adhesins, and secretion and iron-acquisition systems is a fundamental part of understanding infectious disease mechanisms. In contrast, little is known about the contribution of bacterial metabolism to infectious disease. This study shows that E. coli, which cause most urinary tract infections, utilize peptides as a preferred carbon source in vivo and requires some, but not all, of the central metabolic pathways to infect the urinary tract. Specifically, pathways that can be used to replenish metabolites, known as anaplerotic reactions, are important for uropathogenic E. coli infections. These findings help explain how metabolism can contribute to the ability of bacteria to cause a common infection.
Salmonella enterica comprise a family of pathogenic gram-negative bacteria that have evolved sophisticated virulence mechanisms to enter non-phagocytic cells. The entry event is the result of a carefully orchestrated modulation of Rho family GTPase activity within the host cell, which in turn triggers localized remodeling of the actin cytoskeleton. These cytoskeletal rearrangements drive profuse membrane ruffling and lamellipodial extensions that envelop bacteria and trigger their internalization. This chapter describes a number of methods used to investigate the role of Rho family GTPases during Salmonella/host cell interactions. In particular, we detail a variety of complementary techniques, including affinity pull-down assays and bacterial-induced membrane ruffling and internalization assays to show that Salmonella-induced actin remodeling and entry require the Rho family members Rac and RhoG.
Fundamental to the biology of many bacterial pathogens are bacterial proteins with the capacity to modulate host cellular functions. These bacterial proteins are delivered to the host’s molecular targets by a great diversity of mechanisms of varying complexity. The different delivery mechanisms are adapted to the specific biology of the pathogen. Here we focus our attention on a recently described delivery pathway adapted to the biology of an intracellular pathogen, in which an exotoxin is delivered from an intracellular location to its molecular target through autocrine and paracrine pathways.
Campylobacter jejuni is one of the major causes of infectious diarrhea world-wide, although relatively little is know about its mechanisms of pathogenicity. This bacterium can gain entry into intestinal epithelial cells, which is thought to be important for its ability to persistently infect and cause disease. We found that C. jejuni is able to survive within intestinal epithelial cells. However, recovery of intracellular bacteria required pre-culturing under oxygen-limiting conditions, suggesting that C. jejuni undergoes significant physiological changes within the intracellular environment. We also found that in epithelial cells the C. jejuni–containing vacuole deviates from the canonical endocytic pathway immediately after a unique caveolae-dependent entry pathway, thus avoiding delivery into lysosomes. In contrast, in macrophages, C. jejuni is delivered to lysosomes and consequently is rapidly killed. Taken together, these studies indicate that C. jejuni has evolved specific adaptations to survive within host cells.
Campylobacter jejuni is one of the most common causes of food-borne illness in the United States and a major cause of diarrheal disease throughout the world. After infection through the oral route, this bacterium invades the cells of the intestinal epithelium, a property that is important for its ability to cause disease. Usually, bacteria and other material entering the cell move to compartments called lysosomes, where an acidic mix of enzymes breaks it down. This study shows that C. jejuni can survive within intestinal epithelial cells by avoiding delivery to lysosomes. In contrast, in macrophages, which are specialized cells with the capacity to engulf and kill bacteria, C. jejuni cannot avoid delivery into lysosomes and consequently is rapidly killed. These studies help explain an important virulence attribute of C. jejuni.
Campylobacter jejuni is a major worldwide cause of enteric illnesses. Adult immunocompetent mice are not susceptible to C. jejuni infection. However, we show here that mice deficient in the adaptor protein myeloid differentiation factor 88 (MyD88), which is required for signaling through most Toll-like receptors, can be stably colonized by C. jejuni but not by isogenic derivatives carrying mutations in known virulence genes. We also found that Nramp1 deficiency increases the mouse susceptibility to C. jejuni infection when administered systemically. These results indicate that MyD88-deficient mice could be a useful model to study C. jejuni colonization and reveal a potential role for Nramp1 in the control of this bacterial pathogen.
Type III secretion systems (TTSSs) mediate translocation of virulence factors into host cells. We report the 17-angstrom resolution structures of a central component of Salmonella typhimurium TTSS, the needle complex, and its assembly precursor, the bacterial envelope–anchored base. Both the base and the fully assembled needle complex adopted multiple oligomeric states in vivo, and needle assembly was accompanied by recruitment of the protein PrgJ as a structural component of the base. Moreover, conformational changes during needle assembly created scaffolds for anchoring both PrgJ and the needle substructure and may provide the basis for substrate-specificity switching during type III secretion.