Characterization of the integrated filamentous phage Pf5 and its involvement in small-colony formation

Bacteriophages play an important role in bacterial virulence and phenotypic variation. It has been shown that filamentous bacteriophage Pf4 of Pseudomonas aeruginosa strain PAO1 mediates the formation of small-colony variants (SCVs) in biofilms. This morphology type is associated with parameters of poor lung function in cystic fibrosis patients, and SCVs are often more resistant to antibiotics than wild-type cells. P. aeruginosa strain PA14 also contains a Pf1-like filamentous prophage, which is designated Pf5, and is highly homologous to Pf4. Since P. aeruginosa PA14 produces SCVs very efficiently in biofilms grown in static cultures, the role of Pf5 in SCV formation under these conditions was investigated. The presence of the Pf5 replicative form in total DNA from SCVs and wild-type cells was detected, but it was not possible to detect the Pf5 major coat protein by immunoblot analysis in PA14 SCV cultures. This suggests that the Pf5 filamentous phage is not present at high densities in the PA14 SCVs. Consistent with these results, we were unable to detect coaB expression in SCV cultures and SCV colonies. The SCV variants formed under static conditions were not linked to Pf5 phage activity, since Pf5 insertion mutants with decreased or no production of the Pf5 RF produced SCVs as efficiently as the wild-type strain. Finally, analysis of 48 clinical P. aeruginosa isolates showed no association between the presence of Pf1-like filamentous phages and the ability to form SCVs under static conditions; this suggests that filamentous phages are generally not involved in the emergence of P. aeruginosa SCVs

Trehalose Biosynthesis Promotes Pseudomonas aeruginosa Pathogenicity in Plants

Pseudomonas aeruginosa strain PA14 is a multi-host pathogen that infects plants, nematodes, insects, and vertebrates. Many PA14 factors are required for virulence in more than one of these hosts. Noting that plants have a fundamentally different cellular architecture from animals, we sought to identify PA14 factors that are specifically required for plant pathogenesis. We show that synthesis by PA14 of the disaccharide trehalose is required for pathogenesis in Arabidopsis, but not in nematodes, insects, or mice. In-frame deletion of two closely-linked predicted trehalose biosynthetic operons, treYZ and treS, decreased growth in Arabidopsis leaves about 50 fold. Exogenously co-inoculated trehalose, ammonium, or nitrate, but not glucose, sulfate, or phosphate suppressed the phenotype of the double ΔtreYZΔtreS mutant. Exogenous trehalose or ammonium nitrate does not suppress the growth defect of the double ΔtreYZΔtreS mutant by suppressing the plant defense response. Trehalose also does not function intracellularly in P. aeruginosa to ameliorate a variety of stresses, but most likely functions extracellularly, because wild-type PA14 rescued the in vivo growth defect of the ΔtreYZΔtreS in trans. Surprisingly, the growth defect of the double ΔtreYZΔtreS double mutant was suppressed by various Arabidopsis cell wall mutants that affect xyloglucan synthesis, including an xxt1xxt2 double mutant that completely lacks xyloglucan, even though xyloglucan mutants are not more susceptible to pathogens and respond like wild-type plants to immune elicitors. An explanation of our data is that trehalose functions to promote the acquisition of nitrogen-containing nutrients in a process that involves the xyloglucan component of the plant cell wall, thereby allowing P. aeruginosa to replicate in the intercellular spaces in a leaf. This work shows how P. aeruginosa, a multi-host opportunistic pathogen, has repurposed a highly conserved “house-keeping” anabolic pathway (trehalose biosynthesis) as a potent virulence factor that allows it to replicate in the intercellular environment of a leaf

Use of the Galleria mellonella Caterpillar as a Model Host To Study the Role of the Type III Secretion System in Pseudomonas aeruginosa Pathogenesis

Nonvertebrate model hosts represent valuable tools for the study of host-pathogen interactions because they facilitate the identification of bacterial virulence factors and allow the discovery of novel components involved in host innate immune responses. In this report, we determined that the greater wax moth caterpillar Galleria mellonella is a convenient nonmammalian model host for study of the role of the type III secretion system (TTSS) in Pseudomonas aeruginosa pathogenesis. Based on the observation that a mutation in the TTSS pscD gene of P. aeruginosa strain PA14 resulted in a highly attenuated virulence phenotype in G. mellonella, we examined the roles of the four known effector proteins of P. aeruginosa (ExoS, ExoT, ExoU, and ExoY) in wax moth killing. We determined that in P. aeruginosa strain PA14, only ExoT and ExoU play a significant role in G. mellonella killing. Strain PA14 lacks the coding sequence for the ExoS effector protein and does not seem to express ExoY. Moreover, using ΔexoU ΔexoY, ΔexoT ΔexoY, and ΔexoT ΔexoU double mutants, we determined that individual translocation of either ExoT or ExoU is sufficient to obtain nearly wild-type levels of G. mellonella killing. On the other hand, data obtained with a ΔexoT ΔexoU ΔexoY triple mutant and a ΔpscD mutant suggested that additional, as-yet-unidentified P. aeruginosa components of type III secretion are involved in virulence in G. mellonella. A high level of correlation between the results obtained in the G. mellonella model and the results of cytopathology assays performed with a mammalian tissue culture system validated the use of G. mellonella for the study of the P. aeruginosa TTSS

Characterization of the integrated filamentous phage Pf5 and its involvement in small-colony formation

Bacteriophages play an important role in bacterial virulence and phenotypic variation. It has been shown that filamentous bacteriophage Pf4 of Pseudomonas aeruginosa strain PAO1 mediates the formation of small-colony variants (SCVs) in biofilms. This morphology type is associated with parameters of poor lung function in cystic fibrosis patients, and SCVs are often more resistant to antibiotics than wild-type cells. P. aeruginosa strain PA14 also contains a Pf1-like filamentous prophage, which is designated Pf5, and is highly homologous to Pf4. Since P. aeruginosa PA14 produces SCVs very efficiently in biofilms grown in static cultures, the role of Pf5 in SCV formation under these conditions was investigated. The presence of the Pf5 replicative form in total DNA from SCVs and wild-type cells was detected, but it was not possible to detect the Pf5 major coat protein by immunoblot analysis in PA14 SCV cultures. This suggests that the Pf5 filamentous phage is not present at high densities in the PA14 SCVs. Consistent with these results, we were unable to detect coaB expression in SCV cultures and SCV colonies. The SCV variants formed under static conditions were not linked to Pf5 phage activity, since Pf5 insertion mutants with decreased or no production of the Pf5 RF produced SCVs as efficiently as the wild-type strain. Finally, analysis of 48 clinical P. aeruginosa isolates showed no association between the presence of Pf1-like filamentous phages and the ability to form SCVs under static conditions; this suggests that filamentous phages are generally not involved in the emergence of P. aeruginosa SCV

Quorum‐sensing regulator RhlR but not its autoinducer RhlI enables Pseudomonas to evade opsonization

International audienceWhen Drosophila melanogaster feeds on Pseudomonas aeruginosa, some bacteria cross the intestinal barrier and eventually proliferate in the hemocoel. This process is limited by hemocytes through phagocytosis. P. aeruginosa requires the quorum-sensing regulator RhlR to elude the cellular immune response of the fly. RhlI synthesizes the autoinducer signal that activates RhlR. Here, we show that rhlI mutants are unexpectedly more virulent than rhlR mutants, both in fly and in nematode intestinal infection models, suggesting that RhlR has RhlI-independent functions. We also report that RhlR protects P. aeruginosa from opsonization mediated by the Drosophila thioester-containing protein 4 (Tep4). RhlR mutant bacteria show higher levels of Tep4-mediated opsonization, as compared to rhlI mutants, which prevents lethal bacteremia in the Drosophila hemocoel. In contrast, in a septic model of infection, in which bacteria are introduced directly into the hemocoel, Tep4 mutant flies are more resistant to wild-type P. aeruginosa, but not to the rhlR mutant. Thus, depending on the infection route, the Tep4 opsonin can either be protective or detrimental to host defense

The Great ESKAPE: Exploring the Crossroads of Bile and Antibiotic Resistance in Bacterial Pathogens

Throughout the course of infection, many pathogens encounter bactericidal conditions that threaten the viability of the bacteria and impede the establishment of infection. Bile is one of the most innately bactericidal compounds present in humans, functioning to reduce the bacterial burden in the gastrointestinal tract while also aiding in digestion. It is becoming increasingly apparent that pathogens successfully resist the bactericidal conditions of bile, including bacteria that do not normally cause gastrointestinal infections. This review highlights the ability of , , , (ESKAPE), and other enteric pathogens to resist bile and how these interactions can impact the sensitivity of bacteria to various antimicrobial agents. Given that pathogen exposure to bile is an essential component to gastrointestinal transit that cannot be avoided, understanding how bile resistance mechanisms align with antimicrobial resistance is vital to our ability to develop new, successful therapeutics in an age of widespread and increasing antimicrobial resistance