Killing of Caenorhabditis elegans by Pseudomonas aeruginosa used to model mammalian bacterial pathogenesis

We show that a single clinical isolate of the human opportunistic pathogen Pseudomonas aeruginosa (strain PA14), which previously was shown to be pathogenic in mice and plants, also kills Caenorhabditis elegans. The rate of PA14-mediated killing of C. elegans depends on the composition of the agar medium on which PA14 is grown. When PA14 is grown on minimal medium, killing occurs over the course of several days and is referred to as “slow” killing. When PA14 is grown on high-osmolarity medium, killing occurs over the course of several hours and is referred to as “fast” killing. Several lines of evidence, including the fact that heat-killed bacteria are still capable of fast but not slow killing of C. elegans, indicate that fast and slow killing occur by distinct mechanisms. Slow killing involves an infection-like process and correlates with the accumulation of PA14 within worm intestines. Among 10 PA14 virulence-related mutants that had been shown previously to affect pathogenicity in plants and mice, 6 were less effective in killing C. elegans under both fast- and slow-killing conditions, indicating a high degree of commonalty among the P. aeruginosa factors required for pathogenicity in disparate eukaryotic hosts. Thus, we show that a C. elegans pathogenicity model that is genetically tractable from the perspectives of both host and pathogen can be used to model mammalian bacterial pathogenesis.     

Mitophagy confers resistance to siderophore-mediated killing by Pseudomonas aeruginosa

In the arms race of bacterial pathogenesis, bacteria produce an array of toxins and virulence factors that disrupt core host processes. Hosts mitigate the ensuing damage by responding with immune countermeasures. The iron-binding siderophore pyoverdin is a key virulence mediator of the human pathogen Pseudomonas aeruginosa, but its pathogenic mechanism has not been established. Here we demonstrate that pyoverdin enters Caenorhabditis elegans and that it is sufficient to mediate host killing. Moreover, we show that iron chelation disrupts mitochondrial homeostasis and triggers mitophagy both in C. elegans and mammalian cells. Finally, we show that mitophagy provides protection both against the extracellular pathogen P. aeruginosa and to treatment with a xenobiotic chelator, phenanthroline, in C. elegans. Although autophagic machinery has been shown to target intracellular bacteria for degradation (a process known as xenophagy), our report establishes a role for authentic mitochondrial autophagy in the innate immune defense against P. aeruginosa.Iron is an essential trace element used by a wide range of redox enzymes in bacteria, archaea, and eukaryotes. The requirement for iron has created an ongoing struggle between hosts and pathogens as they vie for control of this nutrient. While free iron is already stringently limited in the bloodstream of mammalian hosts, bacterial infection triggers an innate immune response that sequesters iron even further, serving as a mechanism of limiting microbial proliferation (1). Invasive microorganisms, in turn, synthesize and excrete siderophores, soluble extracellular molecules that tightly bind and help solubilize iron found in the environment. In addition, siderophores are important for acquiring iron from host iron storage proteins and the extracellular milieu, which facilitates microbial growth in this specific niche (2). Siderophores are key virulence factors in many pathosystems, including infection with Pseudomonas aeruginosa (1–4). For example, mutants of P. aeruginosa with compromised pyoverdin biosynthesis exhibit attenuated pathogenesis in both C. elegans and in mice (3–5); despite this, the virulence mechanism(s) of siderophores remains unknown. Similarly, little is known about how hosts defend themselves against siderophore exposure and subsequent loss of iron; the notable exception being secretion of a siderocalin, a protein that binds siderophores and minimizes their activity (6). Given the importance of siderophores as virulence determinants, greater insight into this defense process is needed.P. aeruginosa is a key human nosocomial pathogen, responsible for ∼10% of hospital acquired infections and is frequently associated with adverse medical outcomes that include amputation, removal of medical devices, and death (7). P. aeruginosa also infects C. elegans, showing diverse modes of pathogenesis that are partially dependent upon the medium in which the nematodes are exposed, including intestinal infection on agar (where host death is contingent upon quorum-sensing) and a lethal intoxication in liquid that is dependent upon the P. aeruginosa siderophore pyoverdin (reviewed in ref. 8).     

Insights into Campylobacter jejuni colonization of the mammalian intestinal tract using a novel mouse model of infection

A lack of relevant disease models for Campylobacter jejuni has long been an obstacle to research into this common enteric pathogen. We recently published that mice deficient in Single IgG Interleukin-1 related receptor (SIGIRR), a repressor of MyD88-dependent innate immune signaling, were highly susceptible to enteric infection by murine bacterial pathogens. Subsequently, we successfully employed these mice as an animal model for the human pathogen C. jejuni and gained substantial new insights into infection by this pathogen. The infected mice developed significant intestinal inflammation, primarily via TLR4 stimulation. Furthermore, the resulting gastroenteritis was dependent on C. jejuni pathogenesis as bacterial strains suffering mutations in key virulence factors were attenuated in causing disease. The ability to infect SIGIRR-deficient mice with C. jejuni sheds new light onto how these bacteria colonize the mucus layer of the intestinal tract, invade epithelial cells, and raises new prospects for studying the virulence strategies and pathogenesis of C. jejuni.     

Pseudomonas aeruginosa killing of Caenorhabditis elegans used to identify P. aeruginosa virulence factors

We reported recently that the human opportunistic pathogen Pseudomonas aeruginosa strain PA14 kills Caenorhabditis elegans and that many P. aeruginosa virulence factors (genes) required for maximum virulence in mouse pathogenicity are also required for maximum killing of C. elegans. Here we report that among eight P. aeruginosa PA14 TnphoA mutants isolated that exhibited reduced killing of C. elegans, at least five also exhibited reduced virulence in mice. Three of the TnphoA mutants corresponded to the known virulence-related genes lasR, gacA, and lemA. Three of the mutants corresponded to known genes (aefA from Escherichia coli, pstP from Azotobacter vinelandii, and mtrR from Neisseria gonorrhoeae) that had not been shown previously to play a role in pathogenesis, and two of the mutants contained TnphoA inserted into novel sequences. These data indicate that the killing of C. elegans by P. aeruginosa can be exploited to identify novel P. aeruginosa virulence factors important for mammalian pathogenesis.     

Pseudomonas aeruginosa: Host defence in lung diseases

Lung infections caused by the opportunistic pathogen Pseudomonas aeruginosa can present as a spectrum of clinical entities from a rapidly fatal pneumonia in a neutropenic patient to a multi‐decade bronchitis in patients with cystic fibrosis. P. aeruginosa is ubiquitous in our environment, and one of the most versatile pathogens studied, capable of infecting a number of diverse life forms and surviving harsh environmental factors. It is also able to quickly adapt to new environments, including the lung, where it orchestrates virulence factors to acquire necessary nutrients, and if necessary, turn them off to prevent immune recognition. Despite these capabilities, P. aeruginosa rarely infects healthy human lungs. This is secondary to a highly evolved host defence mechanism that efficiently removes inhaled or aspirated pseudomonads. Many arms of the respiratory host defence have been elucidated using P. aeruginosa as a model pathogen. Human infections with P. aeruginosa have demonstrated the importance of the mechanical barrier functions including mucus clearance, and the innate immune system, including the critical role of the neutrophilic response. As more models of persistent or biofilm P. aeruginosa infections are developed, the role of the adaptive immune response will likely become more evident. Understanding the pathogenesis of P. aeruginosa, and the respiratory host defence response to it has, and will continue to, lead to novel therapeutic strategies to help patients.     

Surface attachment induces Pseudomonas aeruginosa virulence

Pseudomonas aeruginosa infects every type of host that has been examined by deploying multiple virulence factors. Previous studies of virulence regulation have largely focused on chemical cues, but P. aeruginosa may also respond to mechanical cues. Using a rapid imaging-based virulence assay, we demonstrate that P. aeruginosa activates virulence in response to attachment to a range of chemically distinct surfaces, suggesting that this bacterial species responds to mechanical properties of its substrates. Surface-activated virulence requires quorum sensing, but activating quorum sensing does not induce virulence without surface attachment. The activation of virulence by surfaces also requires the surface-exposed protein PilY1, which has a domain homologous to a eukaryotic mechanosensor. Specific mutation of the putative PilY1 mechanosensory domain is sufficient to induce virulence in non–surface-attached cells, suggesting that PilY1 mediates surface mechanotransduction. Triggering virulence only when cells are both at high density and attached to a surface—two host-nonspecific cues—explains how P. aeruginosa precisely regulates virulence while maintaining broad host specificity.The bacterium Pseudomonas aeruginosa is a metabolically versatile pathogen that inhabits diverse environments and infects a remarkable range of hosts, including mammals, insects, worms, amoeba, fungi, and other bacteria. P. aeruginosa produces a large number of secreted and cell-associated virulence factors that are redundant and multifactorial (1, 2). Many of P. aeruginosa’s virulence factors—including pyocyanin, elastase, and hydrogen cyanide—are host-nonspecific (3–5), bolstering the ability of P. aeruginosa to attack a large range of hosts. Although many of the virulence factors in P. aeruginosa have been identified, the cues that regulate their activity are less understood. Because many of the virulence factors are host-nonspecific, we explored whether virulence in P. aeruginosa is regulated by host-nonspecific cues.Host cell membranes and cell surfaces are the first line of defense against bacterial toxins and invasion. P. aeruginosa attaches to host cell surfaces early during the infection process. The presence of a surface could thus act as a cue for P. aeruginosa, signaling the presence of a host. Surface attachment is also a critical initial step that enables the establishment of biofilms (6–8). Although biofilms are clearly important for pathogenesis, it remains unclear whether they directly promote host cell killing or mediate other important processes such as long-term colonization.One host-nonspecific cue that could regulate virulence is the mechanical force that bacteria experience upon surface attachment. P. aeruginosa performs surface-associated behaviors (7, 8) such as swarming and twitching (9, 10), but it remains unclear whether P. aeruginosa senses the chemical or mechanical properties of surfaces. There is precedence for mechanotransduction in eukaryotes, in which surface substrate recognition is an important regulator of development and behavior (11). In prokaryotes, surface mechanical forces affect the binding affinity of cells to substrates (12, 13) and alter the rotation of flagella (14, 15). However, the effects of mechanical forces on cell behaviors other than motility are not understood, and the regulation of virulence by mechanical cues has not been explored.Here, we show that attachment to surfaces induces P. aeruginosa to become virulent. Virulence is activated on a variety of chemically distinct abiotic and host surfaces, suggesting that mechanical cues associated with surface attachment activate virulence. We identify PilY1 as a key mediator of surface-activated virulence. PilY1 is a cell-surface–exposed protein that regulates a number of surface-associated behaviors and contains a mechanically sensitive von Willebrand Factor A (VWFa) domain. Although P. aeruginosa lacking PilY1 cannot activate virulence upon surface contact, bacteria with a specific deletion of the VWFa domain hyperactivate virulence, even in the absence of surface contact. Together, our results suggest that cells detect mechanical cues associated with surface attachment through a mechanosensitive pathway that requires the PilY1 protein. We suggest that detecting mechanical cues associated with surface attachment enables P. aeruginosa to induce virulence toward a broad range of hosts without relying upon chemical recognition of any specific host factor.     

Community surveillance enhances Pseudomonas aeruginosa virulence during polymicrobial infection

Most infections result from colonization by more than one microbe. Within such polymicrobial infections, microbes often display synergistic interactions that result in increased disease severity. Although many clinical studies have documented the occurrence of synergy in polymicrobial infections, little is known about the underlying molecular mechanisms. A prominent pathogen in many polymicrobial infections is Pseudomonas aeruginosa, a Gram-negative bacterium that displays enhanced virulence during coculture with Gram-positive bacteria. In this study we discovered that during coinfection, P. aeruginosa uses peptidoglycan shed by Gram-positive bacteria as a cue to stimulate production of multiple extracellular factors that possess lytic activity against prokaryotic and eukaryotic cells. Consequently, P. aeruginosa displays enhanced virulence in a Drosophila model of infection when cocultured with Gram-positive bacteria. Inactivation of a gene (PA0601) required for peptidoglycan sensing mitigated this phenotype. Using Drosophila and murine models of infection, we also show that peptidoglycan sensing results in P. aeruginosa-mediated reduction in the Gram-positive flora in the infection site. Our data suggest that P. aeruginosa has evolved a mechanism to survey the microbial community and respond to Gram-positive produced peptidoglycan through production of antimicrobials and toxins that not only modify the composition of the community but also enhance host killing. Additionally, our results suggest that therapeutic strategies targeting Gram-positive bacteria might be a viable approach for reducing the severity of P. aeruginosa polymicrobial infections.     

Insights into Campylobacter jejuni colonization of the mammalian intestinal tract using a novel mouse model of infection

A lack of relevant disease models for Campylobacter jejuni has long been an obstacle to research into this common enteric pathogen. We recently published that mice deficient in Single IgG Interleukin-1 related receptor (SIGIRR), a repressor of MyD88-dependent innate immune signaling, were highly susceptible to enteric infection by murine bacterial pathogens. Subsequently, we successfully employed these mice as an animal model for the human pathogen C. jejuni and gained substantial new insights into infection by this pathogen. The infected mice developed significant intestinal inflammation, primarily via TLR4 stimulation. Furthermore, the resulting gastroenteritis was dependent on C. jejuni pathogenesis as bacterial strains suffering mutations in key virulence factors were attenuated in causing disease. The ability to infect SIGIRR-deficient mice with C. jejuni sheds new light onto how these bacteria colonize the mucus layer of the intestinal tract, invade epithelial cells, and raises new prospects for studying the virulence strategies and pathogenesis of C. jejuni.     

Murine solid tumours as a novel model to study bacterial biofilm formation in vivo

Bacteria of many species are able to invade and colonize solid tumours in mice. We have focused on Salmonella enterica serovar Typhimurium. Detailed analysis revealed that such tumour‐invading Salmonella form biofilms, thus providing a versatile in vivo test system for studying bacterial phenotypes and host–pathogen interactions. It appears that biofilm formation by S. typhimurium is induced as a defence against the immune system of the host, and in particular against neutrophils. Further, we extended our work to the clinically more relevant biofilm infection by Pseudomonas aeruginosa. The induction of P. aeruginosa biofilms in neoplastic tissue appears to be elicited as a reaction against the immune system. Reconstitution experiments reveal that T cells are responsible for biofilm induction. Isogenic mutants that are no longer able to form biofilms can be used for comparison studies to determine antimicrobial resistance, especially therapeutic efficacy against P. aeruginosa located in biofilms.     

Virulence and stress-related periplasmic protein (VisP) in bacterial/host associations

Gram-negative bacteria have an outer membrane containing LPS. LPS is constituted of an oligosaccharide portion and a lipid-A moiety that embeds this molecule within the outer membrane. LPS is a pathogen-associated molecular pattern, and several pathogens modify their lipid-A as a stealth strategy to avoid recognition by the innate immune system and gain resistance to host factors that disrupt the bacterial cell envelope. An essential feature of Salmonella enterica Typhimurium pathogenesis is its ability to replicate within vacuoles in professional macrophages. S. Typhimurium modifies its lipid-A by hydroxylation by the Fe2+/α-ketoglutarate-dependent dioxygenase enzyme (LpxO). Here, we show that a periplasmic protein of the bacterial oligonucleotide/oligosaccharide-binding fold family, herein named virulence and stress-related periplasmic protein (VisP), on binding to the sugar moiety of peptidoglycan interacts with LpxO. This interaction inhibits LpxO function, leading to decreased LpxO-dependent lipid-A modifications and increasing resistance to stressors within the vacuole environment during intramacrophage replication promoting systemic disease. Consequently, ΔvisP is avirulent in systemic murine infections, where VisP acts through LpxO. Several Gram-negative pathogens harbor both VisP and LpxO, suggesting that this VisP-LpxO mechanism of lipid-A modifications has broader implications in bacterial pathogenesis. Bacterial species devoid of LpxO (e.g., Escherichia coli) have no lipid-A phenotypes associated with the lack of VisP; however, VisP also controls LpxO-independent phenotypes. VisP and LpxO act independently in the S. Typhimurium murine colitis model, with both mutants being attenuated for diverging reasons; ΔvisP is less resistant to cationic antimicrobial peptides, whereas ΔlpxO is deficient for epithelial cell invasion. VisP converges bacterial cell wall homeostasis, stress responses, and pathogenicity.     

Pathogen-host interactions in Pseudomonas aeruginosa pneumonia

Pseudomonas aeruginosa is an important pathogen causing a wide range of acute and chronic infections. P. aeruginosa rarely causes infection in the normal host, but is an efficient opportunistic pathogen causing serious infections in patients who are mechanically ventilated, individuals who are immunocompromised, and patients with malignancies or HIV infection. Among these risk groups, the most vulnerable hosts are neutropenic and patients who are mechanically ventilated. In addition, P. aeruginosa is the most prevalent chronic infection contributing to the pathogenesis of cystic fibrosis. Because of the ubiquitous nature of P. aeruginosa and its ability to develop resistance to antibiotics, it continues to be problematic from a treatment perspective. The pathogenicity of P. aeruginosa is largely caused by multiple bacterial virulence factors and genetic flexibility enabling it to survive in varied environments. Lung injury associated with P. aeruginosa infection results from both the direct destructive effects of the organism on the lung parenchyma and exuberant host immune responses. This article focuses on the major bacterial virulence factors and important aspects of the host immunity that are involved in the pathogenesis of serious P. aeruginosa infection. In addition to antibiotic therapy, strategies directed toward enhancing host defense and/or limiting excessive inflammation could be important to improve outcome in P. aeruginosa lung infections.     

The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae pv. tomato DC3000

We report the complete genome sequence of the model bacterial pathogen Pseudomonas syringae pathovar tomato DC3000 (DC3000), which is pathogenic on tomato and Arabidopsis thaliana. The DC3000 genome (6.5 megabases) contains a circular chromosome and two plasmids, which collectively encode 5,763 ORFs. We identified 298 established and putative virulence genes, including several clusters of genes encoding 31 confirmed and 19 predicted type III secretion system effector proteins. Many of the virulence genes were members of paralogous families and also were proximal to mobile elements, which collectively comprise 7% of the DC3000 genome. The bacterium possesses a large repertoire of transporters for the acquisition of nutrients, particularly sugars, as well as genes implicated in attachment to plant surfaces. Over 12% of the genes are dedicated to regulation, which may reflect the need for rapid adaptation to the diverse environments encountered during epiphytic growth and pathogenesis. Comparative analyses confirmed a high degree of similarity with two sequenced pseudomonads, Pseudomonas putida and Pseudomonas aeruginosa, yet revealed 1,159 genes unique to DC3000, of which 811 lack a known function.     

Application of Laboratory Research in UTI

With the development of molecular biology, new techniques in urinary tract infection (UTI) research have been introduced. This article reports on study models, molecular techniques and genetic engineering presently used in studies on UTI pathogenesis. Recent research is reviewed, and molecular bacterial virulence mechanisms and the role of inflammation in the antibacterial defenses are discussed. Pivotal studies are presented in more detail to demonstrate the use of the new methods: the in vivo investigation of bacterial virulence factors and host interaction in a human colonization protocol; the experimental mouse UTI model where mice strains differing in genetics demonstrate selective dysfunctions; the search for human polymorphism explaining suceptibility to infectious diseases.     

Dissecting virulence: systematic and functional analyses of a pathogenicity island

Bacterial pathogenicity islands (PAI) often encode both effector molecules responsible for disease and secretion systems that deliver these effectors to host cells. Human enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli, and the mouse pathogen Citrobacter rodentium (CR) possess the locus of enterocyte effacement (LEE) PAI. We systematically mutagenized all 41 CR LEE genes and functionally characterized these mutants in vitro and in a murine infection model. We identified 33 virulence factors, including two virulence regulators and a hierarchical switch for type III secretion. In addition, 7 potential type III effectors encoded outside the LEE were identified by using a proteomics approach. These non-LEE effectors are encoded by three uncharacterized PAIs in EHEC O157, suggesting that these PAIs act cooperatively with the LEE in pathogenesis. Our findings provide significant insights into bacterial virulence mechanisms and disease.     

Human platelet gel supernatant inactivates opportunistic wound pathogens on skin

Activation of human platelets produces a gel-like substance referred to as platelet rich plasma or platelet gel. Platelet gel is used clinically to promote wound healing; it also exhibits antimicrobial properties that may aid in the healing of infected wounds. The purpose of this study was to quantify the efficacy of human platelet gel against the opportunistic bacterial wound pathogens Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus on skin. These opportunistic pathogens may exhibit extensive antibiotic resistance, necessitating the development of alternative treatment options. The antimicrobial efficacy of platelet gel supernatants was quantified using an in vitro broth dilution assay, an ex vivo inoculated skin assay, and in an in vivo skin decontamination assay. Human platelet gel supernatants were highly bactericidal against A. baumannii and moderately but significantly bactericidal against S. aureus in vitro and in the ex vivo skin model. P. aeruginosa was not inactivated in vitro; a low but significant inactivation level was observed ex vivo. These supernatants were quite effective at inactivating a model organism on skin in vivo. These results suggest application of platelet gel has potential clinical applicability, not only in the acceleration of wound healing, but also against relevant bacteria causing wound infections.