High-throughput sequencing of transposon (Tn) libraries created within entire genomes identifies and quantifies the contribution of individual genes and operons to the fitness of organisms in ...different environments. We used insertion-sequencing (INSeq) to analyze the contribution to fitness of all non-essential genes in the chromosome of Pseudomonas aeruginosa strain PA14 based on a library of ∼300,000 individual Tn insertions. In vitro growth in LB provided a baseline for comparison with the survival of the Tn insertion strains following 6 days of colonization of the murine gastrointestinal tract as well as a comparison with Tn-inserts subsequently able to systemically disseminate to the spleen following induction of neutropenia. Sequencing was performed following DNA extraction from the recovered bacteria, digestion with the MmeI restriction enzyme that hydrolyzes DNA 16 bp away from the end of the Tn insert, and fractionation into oligonucleotides of 1,200-1,500 bp that were prepared for high-throughput sequencing. Changes in frequency of Tn inserts into the P. aeruginosa genome were used to quantify in vivo fitness resulting from loss of a gene. 636 genes had <10 sequencing reads in LB, thus defined as unable to grow in this medium. During in vivo infection there were major losses of strains with Tn inserts in almost all known virulence factors, as well as respiration, energy utilization, ion pumps, nutritional genes and prophages. Many new candidates for virulence factors were also identified. There were consistent changes in the recovery of Tn inserts in genes within most operons and Tn insertions into some genes enhanced in vivo fitness. Strikingly, 90% of the non-essential genes were required for in vivo survival following systemic dissemination during neutropenia. These experiments resulted in the identification of the P. aeruginosa strain PA14 genes necessary for optimal survival in the mucosal and systemic environments of a mammalian host.
Clinical isolates of Staphylococcus aureus can express biofilm phenotypes promoted by the major cell wall autolysin and the fibronectin-binding proteins or the icaADBC-encoded polysaccharide ...intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG). Biofilm production in methicillin-susceptible S. aureus (MSSA) strains is typically dependent on PIA/PNAG whereas methicillin-resistant isolates express an Atl/FnBP-mediated biofilm phenotype suggesting a relationship between susceptibility to β-lactam antibiotics and biofilm. By introducing the methicillin resistance gene mecA into the PNAG-producing laboratory strain 8325-4 we generated a heterogeneously resistant (HeR) strain, from which a homogeneous, high-level resistant (HoR) derivative was isolated following exposure to oxacillin. The HoR phenotype was associated with a R₆₀₂H substitution in the DHHA1 domain of GdpP, a recently identified c-di-AMP phosphodiesterase with roles in resistance/tolerance to β-lactam antibiotics and cell envelope stress. Transcription of icaADBC and PNAG production were impaired in the 8325-4 HoR derivative, which instead produced a proteinaceous biofilm that was significantly inhibited by antibodies against the mecA-encoded penicillin binding protein 2a (PBP2a). Conversely excision of the SCCmec element in the MRSA strain BH1CC resulted in oxacillin susceptibility and reduced biofilm production, both of which were complemented by mecA alone. Transcriptional activity of the accessory gene regulator locus was also repressed in the 8325-4 HoR strain, which in turn was accompanied by reduced protease production and significantly reduced virulence in a mouse model of device infection. Thus, homogeneous methicillin resistance has the potential to affect agr- and icaADBC-mediated phenotypes, including altered biofilm expression and virulence, which together are consistent with the adaptation of healthcare-associated MRSA strains to the antibiotic-rich hospital environment in which they are frequently responsible for device-related infections in immuno-compromised patients.
Therapeutic intervention in cystic fibrosis (CF) remains a challenge, partly because of the number of organs and tissues affected by the lack of a functional cystic fibrosis transmembrane conductance ...regulator (CFTR) protein. CF was originally regarded primarily as a gastrointestinal (GI) disease because of the failure to thrive and early death from malnutrition in infants with CF. However, successful interventions for the GI manifestations of CF have left chronic lung infections as the primary cause of morbidity and mortality. Despite a complex microbiology within the CF lung, one pathogen, Pseudomonas aeruginosa, remains the critical determinant of pulmonary pathology. Treatment and management of this infection and its associated symptoms are the major targets of extant and developing CF therapies. Understanding the multitude of effects of CFTR on mucosal physiology and susceptibility and progression of chronic lung disease, and how host immune responses fail to adequately control lung infection, will be essential for the development of improved therapies for CF.
Th17 immunity in the gastrointestinal tract is regulated by the intestinal microbiota composition, particularly the presence of segmented filamentous bacteria (sfb), but the role of the intestinal ...microbiota in pulmonary host defense is not well explored. We tested whether altering the gut microbiota by acquiring sfb influences the susceptibility to staphylococcal pneumonia via induction of type 17 immunity. Groups of C57BL/6 mice which differed in their intestinal colonization with sfb were challenged with methicillin-resistant Staphylococcus aureus in an acute lung infection model. Bacterial burdens, bronchoalveolar lavage fluid (BALF) cell counts, cell types, and cytokine levels were compared between mice from different vendors, mice from both vendors after cohousing, mice given sfb orally prior to infection, and mice with and without exogenous interleukin-22 (IL-22) or anti-IL-22 antibodies. Mice lacking sfb developed more severe S. aureus pneumonia than mice colonized with sfb, as indicated by higher bacterial burdens in the lungs, lung inflammation, and mortality. This difference was reduced when sfb-negative mice acquired sfb in their gut microbiota through cohousing with sfb-positive mice or when given sfb orally. Levels of type 17 immune effectors in the lung were higher after infection in sfb-positive mice and increased in sfb-negative mice after acquisition of sfb, as demonstrated by higher levels of IL-22 and larger numbers of IL-22(+) TCRβ(+) cells and neutrophils in BALF. Exogenous IL-22 protected mice from S. aureus pneumonia. The murine gut microbiota, particularly the presence of sfb, promotes pulmonary type 17 immunity and resistance to S. aureus pneumonia, and IL-22 protects against severe pulmonary staphylococcal infection.
Summary A number of different vaccines and several monoclonal antibodies have been developed in the last decades for active and passive vaccination against the Gram-negative opportunistic pathogen ...Pseudomonas aeruginosa. These approaches include vaccine antigens such as lipopolysaccharide, surface polysaccharides, polysaccharide–protein conjugates, flagella, outer membrane proteins, pili, whole formalin-killed P. aeruginosa cells, live-attenuated P. aeruginosa and Salmonella enterica strains expressing P. aeruginosa antigens and DNA sequences. While many of these experimental vaccines and monoclonal antibodies have been tested in preclinical trials, only a few have reached clinical phases and none of these vaccines has obtained market authorization. The purpose of this review is to provide a brief summary of the present state of the development of vaccines and immunotherapies against P. aeruginosa infections. According to the different types of infection caused by P. aeruginosa – localized on mucosal surfaces such as the airways or systemic infection in the blood stream – several potential routes suggesting optimal means to administer the experimental vaccines are presented. Finally, the inherent problem of testing P. aeruginosa candidate vaccines in patient populations is discussed.
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•Antibodies recognise glycans by end-on insertion, bifurcated or groove binding modes.•Antibody specificity is dictated by the diversity of bacterial carbohydrate epitopes.•Bacterial ...carbohydrates can be targeted by protective and non-protective antibodies.
Because of the ongoing increase in antibiotic-resistant microbes, new strategies such as therapeutic antibodies and effective vaccines are required. Bacterial carbohydrates are known to be particularly antigenic, and several monoclonal antibodies that target bacterial polysaccharides have been generated, with more in current development. This review examines the known 3D crystal structures of anti-bacterial antibodies and the structural basis for carbohydrate recognition and explores the potential mechanisms for antibody-dependent bacterial cell death. Understanding the key interactions between an antibody and its polysaccharide target on the surface of bacteria or in biofilms can provide essential information for the development of more specific and effective antibody therapeutics as well as carbohydrate-based vaccines.
Abstract Pseudomonas aeruginosa is one of the most important bacterial pathogens encountered by immunocompromised hosts and patients with cystic fibrosis (CF), and the lipopolysaccharide (LPS) ...elaborated by this organism is a key factor in virulence as well as both innate and acquired host responses to infection. The molecule has a fair degree of heterogeneity in its lipid A and O-antigen structure, and elaborates two different outer-core glycoforms, of which only one is ligated to the O-antigen. A close relatedness between the chemical structures and genes encoding biosynthetic enzymes has been established, with 11 major O-antigen groups identified. The lipid A can be variably penta-, hexa- or hepta-acylated, and these isoforms have differing potencies when activating host innate immunity via binding to Toll-like receptor 4 (TLR4). The O-antigen is a major target for protective immunity as evidenced by numerous animal studies, but attempts, to date, to produce a human vaccine targeting these epitopes have not been successful. Newer strategies employing live attenuated P. aeruginosa , or heterologous attenuated bacteria expressing P. aeruginosa O-antigens are potential means to solve some of the existing problems related to making a P. aeruginosa LPS-specific vaccine. Overall, there is now a large amount of information available about the genes and enzymes needed to produce the P. aeruginosa LPS, detailed chemical structures have been determined for the major O-antigens, and significant biologic and immunologic studies have been conducted to define the role of this molecule in virulence and immunity to P. aeruginosa infection.
The biofilm component poly-
-acetylglucosamine (PNAG) is an important virulence determinant in medical-device-related infections caused by ESKAPE group pathogens including Gram-positive
and ...Gram-negative
. PNAG presentation on bacterial cell surfaces and its accessibility for host interactions are not fully understood. We employed a lectin microarray to examine PNAG surface presentation and interactions on methicillin-sensitive (MSSA) and methicillin-resistant
(MRSA) and a clinical
isolate. Purified PNAG bound to wheatgerm agglutinin (WGA) and succinylated WGA (sWGA) lectins only. PNAG was the main accessible surface component on MSSA but was relatively inaccessible on the
surface, where it modulated the presentation of other surface molecules. Carbohydrate microarrays demonstrated similar specificities of
and
for their most intensely binding carbohydrates, including 3' and 6'sialyllactose, but differences in moderately binding ligands, including blood groups A and B. An
-acetylglucosamine-binding lectin function which binds to PNAG identified on the
cell surface may contribute to biofilm structure and PNAG surface presentation on
. Overall, these data indicated differences in PNAG presentation and accessibility for interactions on Gram-positive and Gram-negative cell surfaces which may play an important role in biofilm-mediated pathogenesis.
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