Characterization of BCAM0224, a Multifunctional Trimeric Autotransporter from the Human Pathogen Burkholderia cenocepacia

Members of the trimeric autotransporter adhesin (TAA) family play a crucial role in adhesion of Gram-negative pathogens to host cells. Moreover, these proteins are multifunctional virulence factors involved in several other biological traits, including invasion into host cells and evasion of the host immune system. In cystic fibrosis epidemic Burkholderia cenocepacia strain J2315, we identified a unique TAA (BCAM0224)-encoding gene, previously described as being implicated in virulence. Here, we characterized this multifunctional protein, trying to establish its role in B. cenocepacia pathogenicity. We show that BCAM0224 occurs on the bacterial surface and adopts a trimeric conformation. Furthermore, we demonstrated that BCAM0224 is needed for earlier stages of biofilm formation and is required for swarming motility. In addition, BCAM0224 plays an important role in evasion of the human innate immune system, providing resistance against the bactericidal activity of serum via the complement classical pathway. Finally, BCAM0224 mediates bacterial adhesion to and invasion of cultured human bronchial epithelial cells. Together, these data reveal the high versatility of the BCAM0224 protein as a virulence factor in the pathogenic bacterium B. cenocepacia.     

Using model systems to unravel host–Pseudomonas aeruginosa interactions

Using model systems in infection biology has led to the discoveries of many pathogen-encoded virulence factors and critical host immune factors to fight pathogenic infections. Studies of the remarkable Pseudomonas aeruginosa bacterium that infects and causes disease in hosts as divergent as humans and plants afford unique opportunities to shed new light on virulence strategies and host defence mechanisms. One of the rationales for using model systems as a discovery tool to characterise bacterial factors driving human infection outcomes is that many P. aeruginosa virulence factors are required for pathogenesis in diverse different hosts. On the other side, many host signalling components, such as the evolutionarily conserved mitogen-activated protein kinases, are involved in immune signalling in a diverse range of hosts. Some model organisms that have less complex immune systems also allow dissection of the direct impacts of innate immunity on host defence without the interference of adaptive immunity. In this review, we start with discussing the occurrence of P. aeruginosa in the environment and the ability of this bacterium to cause disease in various hosts as a natural opportunistic pathogen. We then summarise the use of some model systems to study host defence and P. aeruginosa virulence.     

Host-microbe interactions that shape the pathogenesis of Acinetobacter baumannii infection

Acinetobacter baumannii is an opportunistic pathogen that has emerged as a prevalent source of nosocomial infections, most frequently causing ventilator‐associated pneumonia. The emergence of pan‐drug resistant strains magnifies the problem by reducing viable treatment options and effectively increasing the mortality rate associated with Acinetobacter infections. In light of this rising threat, research on A. baumannii epidemiology, antibiotic resistance, and pathogenesis is accelerating. The recent development of both in vitro and in vivo models has enabled studies probing the host–Acinetobacter interface. Bacterial genetic screens and comparative genomic studies have led to the identification of several A. baumannii virulence factors. Additionally, investigations into host defence mechanisms using animal models or cell culture have provided insight into the innate immune response to infection. This review highlights some of the key attributes of A. baumannii virulence with an emphasis on bacterial interactions with the innate immune system.     

The interface between virulence and host response to the pathogenic fungus Histoplasma capsulatum

Intracellular pathogens have evolved machinery to evade the immune response in order to survive within a host. Histoplasma capsulatum, one of the intracellular pathogens, is a dimorphic fungus that dodges innate and adaptive immunity; it escapes immunity presumably through virulence factors that permit fungal survival and replication within the host. This review discusses immune factors that contribute to the control of H. capsulatum infection, several host-survival mechanisms H. capsulatum uses, and new techniques that have led to the identification of several H. capsulatum virulence factors, which will most likely aid in the discovery of many more.     

DNase Sda1 Allows Invasive M1T1 Group A Streptococcus to Prevent TLR9-Dependent Recognition

Group A Streptococcus (GAS) has developed a broad arsenal of virulence factors that serve to circumvent host defense mechanisms. The virulence factor DNase Sda1 of the hyperinvasive M1T1 GAS clone degrades DNA-based neutrophil extracellular traps allowing GAS to escape extracellular killing. TLR9 is activated by unmethylated CpG-rich bacterial DNA and enhances innate immune resistance. We hypothesized that Sda1 degradation of bacterial DNA could alter TLR9-mediated recognition of GAS by host innate immune cells. We tested this hypothesis using a dual approach: loss and gain of function of DNase in isogenic GAS strains and presence and absence of TLR9 in the host. Either DNA degradation by Sda1 or host deficiency of TLR9 prevented GAS induced IFN-α and TNF-α secretion from murine macrophages and contributed to bacterial survival. Similarly, in a murine necrotizing fasciitis model, IFN-α and TNF-α levels were significantly decreased in wild type mice infected with GAS expressing Sda1, whereas no such Sda1-dependent effect was seen in a TLR9-deficient background. Thus GAS Sda1 suppressed both the TLR9-mediated innate immune response and macrophage bactericidal activity. Our results demonstrate a novel mechanism of bacterial innate immune evasion based on autodegradation of CpG-rich DNA by a bacterial DNase.     

Common themes in microbial pathogenicity revisited.

Bacterial pathogens employ a number of genetic strategies to cause infection and, occasionally, disease in their hosts. Many of these virulence factors and their regulatory elements can be divided into a smaller number of groups based on the conservation of similar mechanisms. These common themes are found throughout bacterial virulence factors. For example, there are only a few general types of toxins, despite a large number of host targets. Similarly, there are only a few conserved ways to build the bacterial pilus and nonpilus adhesins used by pathogens to adhere to host substrates. Bacterial entry into host cells (invasion) is a complex mechanism. However, several common invasion themes exist in diverse microorganisms. Similarly, once inside a host cell, pathogens have a limited number of ways to ensure their survival, whether remaining within a host vacuole or by escaping into the cytoplasm. Avoidance of the host immune defenses is key to the success of a pathogen. Several common themes again are employed, including antigenic variation, camouflage by binding host molecules, and enzymatic degradation of host immune components. Most virulence factors are found on the bacterial surface or secreted into their immediate environment, yet virulence factors operate through a relatively small number of microbial secretion systems. The expression of bacterial pathogenicity is dependent upon complex regulatory circuits. However, pathogens use only a small number of biochemical families to express distinct functional factors at the appropriate time that causes infection. Finally, virulence factors maintained on mobile genetic elements and pathogenicity islands ensure that new strains of pathogens evolve constantly. Comprehension of these common themes in microbial pathogenicity is critical to the understanding and study of bacterial virulence mechanisms and to the development of new "anti-virulence" agents, which are so desperately needed to replace antibiotics.     

Virulence determinants of the human pathogenic fungus Aspergillus fumigatus protect against soil amoeba predation

Filamentous fungi represent classical examples for environmentally acquired human pathogens whose major virulence mechanisms are likely to have emerged long before the appearance of innate immune systems. In natural habitats, amoeba predation could impose a major selection pressure towards the acquisition of virulence attributes. To test this hypothesis, we exploited the amoeba Dictyostelium discoideum to study its interaction with Aspergillus fumigatus, two abundant soil inhabitants for which we found co‐occurrence in various sites. Fungal conidia were efficiently taken up by D. discoideum, but ingestion was higher when conidia were devoid of the green fungal spore pigment dihydroxynaphtalene melanin, in line with earlier results obtained for immune cells. Conidia were able to survive phagocytic processing, and intracellular germination was initiated only after several hours of co‐incubation which eventually led to a lethal disruption of the host cell. Besides phagocytic interactions, both amoeba and fungus secreted cross inhibitory factors which suppressed fungal growth or induced amoeba aggregation with subsequent cell lysis, respectively. On the fungal side, we identified gliotoxin as the major fungal factor killing Dictyostelium, supporting the idea that major virulence attributes, such as escape from phagocytosis and the secretion of mycotoxins are beneficial to escape from environmental predators.     

Virulence of Vibrio harveyi ORM4 towards the European abalone Haliotis tuberculata involves both quorum sensing and a type III secretion system

Environmental Vibrio strains represent a major threat in aquaculture, but the understanding of their virulence mechanisms heavily relies on the transposition of knowledge from human-pathogen vibrios. Here, the genetic bases of the virulence of Vibrio harveyi ORM4 towards the European abalone Haliotis tuberculata were characterized. We demonstrated that luxO, encoding a major regulator of the quorum sensing system, is crucial for the virulence of this strain, and that its deletion leads to a decrease in swimming motility, biofilm formation, and exopolysaccharide production. Furthermore, the biofilm formation by V. harveyi ORM4 was increased by abalone serum, which required LuxO. The absence of LuxO in V. harveyi ORM4 yielded opposite phenotypes compared with other Vibrio species including V. campbellii (still frequently named V. harveyi). In addition, we report a full type III secretion system (T3SS) gene cluster in the V. harveyi ORM4 genome. LuxO was shown to negatively regulate the promoter activity of exsA, encoding the major regulator of the T3SS genes, and the deletion of exsA abolished the virulence of V. harveyi ORM4. These results unveil virulence mechanisms set up by this environmentally important bacterial pathogen and pave the way for a better molecular understanding of the regulation of its pathogenicity.     

Bacterial toxins can inhibit host cell autophagy through cAMP generation

Autophagy plays a significant role in innate and adaptive immune responses to microbial infection. Some pathogenic bacteria have developed strategies to evade killing by host autophagy. These include the use of ‘camouflage’ proteins to block targeting to the autophagy pathway and the use of pore-forming toxins to block autophagosome maturation. However, general inhibition of host autophagy by bacterial pathogens has not been observed to date. Here we demonstrate that bacterial cAMP-elevating toxins from B. anthracis and V. cholera can inhibit host anti-microbial autophagy, including autophagic targeting of S. Typhimurium and latex bead phagosomes. Autophagy inhibition required the cAMP effector protein kinase A. Formation of autophagosomes in response to rapamycin and the endogenous turnover of peroxisomes was also inhibited by cAMP-elevating toxins. These findings demonstrate that cAMP-elevating toxins, representing a large group of bacterial virulence factors, can inhibit host autophagy to suppress immune responses and modulate host cell physiology.     

Adaptive processes of Staphylococcus aureus isolates during the progression from acute to chronic bone and joint infections in patients

Staphylococcus aureus bone and joint infection (BJI) is associated with significant rates of chronicity and relapse. In this study, we investigated how S. aureus is able to adapt to the human environment by comparing isolates from single patients with persisting or relapsing BJIs that were recovered during the initial and recurrent BJI episodes. In vitro and in vivo assays and whole‐genome sequencing analyses revealed that the recurrent isolates induced a reduced inflammatory response, formed more biofilms, persisted longer in the intracellular compartments of host bone cells, were less cytotoxic and induced less mortality in a mouse infection model compared with the initial isolates despite the lack of significant changes at the genomic level. These findings suggest that S. aureus BJI chronicization is associated with an in vivo bacterial phenotypical adaptation that leads to decreased virulence and host immune escape, which is linked to increased intraosteoblastic persistence and biofilm formation.     

Modulation of innate immune responses by Yersinia type III secretion system translocators and effectors

The innate immune system of mammals responds to microbial infection through detection of conserved molecular determinants called ‘pathogen‐associated molecular patterns’ (PAMPs). Pathogens use virulence factors to counteract PAMP‐directed responses. The innate immune system can in turn recognize signals generated by virulence factors, allowing for a heightened response to dangerous pathogens. Many Gram‐negative bacterial pathogens encode type III secretion systems (T3SSs) that translocate effector proteins, subvert PAMP‐directed responses and are critical for infection. A plasmid‐encoded T3SS in the human‐pathogenic Yersinia species translocates seven effectors into infected host cells. Delivery of effectors by the T3SS requires plasma membrane insertion of two translocators, which are thought to form a channel called a translocon. Studies of the Yersinia T3SS have provided key advances in our understanding of how innate immune responses are generated by perturbations in plasma membrane and other signals that result from translocon insertion. Additionally, studies in this system revealed that effectors function to inhibit innateimmune responses resulting from insertion of translocons into plasma membrane. Here, we review these advances with the goal of providing insight into how a T3SS can activate and inhibit innate immune responses, allowing a virulent pathogen to bypass host defences.     

Mechanisms of enterococcal survival in the host

Information on virulence and persistence factors of enterococci as important nosocomial pathogens with ever increasing multiple drug resistance is updated. The importance of the virulent and persistent properties of enterococci in the processes of adhesion to host tissues, invasion, modulation of inflammatory responses, as well as their toxic action on the host body is considered. The pathogenicity factors of enterococci are relevant to the mechanisms of the bacterial survival in the host.     

RhoGTPases and inflammasomes: Guardians of effector-triggered immunity

Pathogens have evolved smart strategies to invade hosts and hijack their immune responses. One such strategy is the targeting of the host RhoGTPases by toxins or virulence factors to hijack the cytoskeleton dynamic and immune processes. In response to this microbial attack, the host has evolved an elegant strategy to monitor the function of virulence factors and toxins by sensing the abnormal activity of RhoGTPases. This innate immune strategy of sensing bacterial effector targeting RhoGTPase appears to be a bona fide example of effector-triggered immunity (ETI). Here, we review recently discovered mechanisms by which the host can sense the activity of these toxins through NOD and NOD-like receptors (NLRs).     

葡萄球菌致病毒力因子酚溶性调节蛋白的研究进展

葡萄球菌是临床常见的革兰氏阳性致病菌,包括表皮葡萄球菌和金黄色葡萄球菌等.随着耐药葡萄球菌的出现,尤其是多药耐药葡萄球菌的传播和蔓延,其感染性疾病的发病率和死亡率逐年升高.葡萄球菌的高致病性与其表达大量毒力因子密切相关.酚溶性调节蛋白(phenol-soluble modulin,PSM)是一组具有广泛溶细胞活性的两亲...     

Glutamate Utilization Couples Oxidative Stress Defense and the Tricarboxylic Acid Cycle in Francisella Phagosomal Escape

Intracellular bacterial pathogens have developed a variety of strategies to avoid degradation by the host innate immune defense mechanisms triggered upon phagocytocis. Upon infection of mammalian host cells, the intracellular pathogen Francisella replicates exclusively in the cytosolic compartment. Hence, its ability to escape rapidly from the phagosomal compartment is critical for its pathogenicity. Here, we show for the first time that a glutamate transporter of Francisella (here designated GadC) is critical for oxidative stress defense in the phagosome, thus impairing intra-macrophage multiplication and virulence in the mouse model. The gadC mutant failed to efficiently neutralize the production of reactive oxygen species. Remarkably, virulence of the gadC mutant was partially restored in mice defective in NADPH oxidase activity. The data presented highlight links between glutamate uptake, oxidative stress defense, the tricarboxylic acid cycle and phagosomal escape. This is the first report establishing the role of an amino acid transporter in the early stage of the Francisella intracellular lifecycle.     

TLR signaling is required for Salmonella typhimurium virulence

Toll-like receptors (TLRs) contribute to host resistance to microbial pathogens and can drive the evolution of virulence mechanisms. We have examined the relationship between host resistance and pathogen virulence using mice with a functional allele of the nramp-1 gene and lacking combinations of TLRs. Mice deficient in both TLR2 and TLR4 were highly susceptible to the intracellular bacterial pathogen Salmonella typhimurium, consistent with reduced innate immune function. However, mice lacking additional TLRs involved in S. typhimurium recognition were less susceptible to infection. In these TLR-deficient cells, bacteria failed to upregulate Salmonella pathogenicity island 2 (SPI-2) genes and did not form a replicative compartment. We demonstrate that TLR signaling enhances the rate of acidification of the Salmonella-containing phagosome, and inhibition of this acidification prevents SPI-2 induction. Our results indicate that S. typhimurium requires cues from the innate immune system to regulate virulence genes necessary for intracellular survival, growth, and systemic infection.     

Lessons from signature-tagged mutagenesis on the infectious mechanisms of pathogenic bacteria

Studies on the genetic basis of bacterial pathogenicity have been undertaken for almost 30 years, but the development of new genetic tools in the past 10 years has considerably increased the number of identified virulence factors. Signature-tagged mutagenesis (STM) is one of the most powerful general genetic approaches, initially developed by David Holden and colleagues in 1995, which has now led to the identification of hundreds of new genes requested for virulence in a broad range of bacterial pathogens. We have chosen to present in this review, the most recent and/or most significant contributions to the understanding of the molecular mechanisms of bacterial pathogenicity among over 40 STM screens published to date. We will first briefly review the principle of the method and its major technical limitations. Then, selected studies will be discussed where genes implicated in various aspects of the infectious process have been identified (including tropism for specific host and/or particular tissues, interactions with host cells, mechanisms of survival and persistence within the host, and the crossing of the blood brain barrier). The examples chosen will cover intracellular as well as extracellular Gram-negative and Gram-positive pathogens.