Application of WGS data for O-specific antigen analysis and <i>in silico </i>serotyping of <i>Pseudomonas aeruginosa </i>isolates

Accurate typing methods are required for efficient infection control. The emergence of whole-genome sequencing (WGS) technologies has enabled the development of genome-based methods applicable for routine typing and surveillance of bacterial pathogens. In this study, we developed the Pseudomonas aeruginosa serotyper (PAst) program, which enabled in silico serotyping of P. aeruginosa isolates using WGS data. PAst has been made publically available as a web service and aptly facilitates high-throughput serotyping analysis. The program overcomes critical issues such as the loss of in vitro typeability often associated with P. aeruginosa isolates from chronic infections and quickly determines the serogroup of an isolate based on the sequence of the O-specific antigen (OSA) gene cluster. Here, PAst analysis of 1,649 genomes resulted in successful serogroup assignments in 99.27% of the cases. This frequency is rarely achievable by conventional serotyping methods. The limited number of nontypeable isolates found using PAst was the result of either a complete absence of OSA genes in the genomes or the artifact of genomic misassembly. With PAst, P. aeruginosa serotype data can be obtained from WGS information alone. PAst is a highly efficient alternative to conventional serotyping methods in relation to outbreak surveillance of serotype O12 and other high-risk clones, while maintaining backward compatibility to historical serotype data

A Bacteriophage-Acquired O-Antigen Polymerase (Wzy_β) from <i>P. aeruginosa </i>Serotype O16 Performs a Varied Mechanism Compared to Its Cognate Wzy_α

Pseudomonas aeruginosa is a Gram-negative bacterium that produces highly varied lipopolysaccharide (LPS) structures. The O antigen (O-Ag) in the LPS is synthesized through the Wzx/Wzy-dependent pathway where lipid-linked O-Ag repeats are polymerized by Wzy. Horizontal-gene transfer has been associated with O-Ag diversity. The O-Ag present on the surface of serotypes O5 and O16, differ in the intra-molecular bonds, α and β, respectively; the latter arose from the action of three genes in a seroconverting unit acquired from bacteriophage D3, including a β-polymerase (Wzyβ). To further our understanding of O-polymerases, the inner membrane (IM) topology of Wzyβ was determined using a dual phoA-lacZα reporter system wherein random 3’ gene truncations were localized to specific loci with respect to the IM by normalized reporter activities as determined through the ratio of alkaline phosphate activity to β-galactosidase activity. The topology of Wzyβ developed through this approach was shown to contain two predominant periplasmic loops, PL3 (containing an RX10G motif) and PL4 (having an O-Ag ligase superfamily motif), associated with inverting glycosyltransferase reaction. Through site-directed mutagenesis and complementation assays, residues Arg254, Arg270, Arg272 and His300 were found to be essential for Wzyβ function. Additionally, like-charge substitutions, R254K and R270K, could not complement the wzyβ knockout, highlighting the essential guanidium side group of Arg residues. The O-Ag ligase domain is conserved among heterologous Wzy proteins that produce β-linked O-Ag repeat units. Taking advantage of the recently obtained whole-genome sequence of serotype O16 a candidate promoter was identified. Wzyβ under its native promoter was integrated in the PAO1 genome, which resulted in simultaneous production of α- and β-linked O-Ag. These observations established that members of Wzy-like family consistently exhibit a dual-periplasmic loops topology, and identifies motifs that are plausible to be involved in enzymatic activities. Based on these results, the phage-derived Wzyβ utilizes a different reaction mechanism in the P. aeruginosa host to avoid self-inhibition during serotype conversion

The Widespread Multidrug-Resistant Serotype O12 <i>Pseudomonas aeruginosa</i> Clone Emerged through Concomitant Horizontal Transfer of Serotype Antigen and Antibiotic Resistance Gene Clusters

The O-specific antigen (OSA) in Pseudomonas aeruginosa lipopolysaccharide is highly varied by sugar identity, side chains, and bond between O-repeats. These differences classified P. aeruginosa into 20 distinct serotypes. In the past few decades, O12 has emerged as the predominant serotype in clinical settings and outbreaks. These serotype O12 isolates exhibit high levels of resistance to various classes of antibiotics. Here, we explore how the P. aeruginosa OSA biosynthesis gene clusters evolve in the population by investigating the association between the phylogenetic relationships among 83 P. aeruginosa strains and their serotypes. While most serotypes were closely linked to the core genome phylogeny, we observed horizontal exchange of OSA biosynthesis genes among phylogenetically distinct P. aeruginosa strains. Specifically, we identified a “serotype island” ranging from 62 kb to 185 kb containing the P. aeruginosa O12 OSA gene cluster, an antibiotic resistance determinant (gyrA(C248T)), and other genes that have been transferred between P. aeruginosa strains with distinct core genome architectures. We showed that these genes were likely acquired from an O12 serotype strain that is closely related to P. aeruginosa PA7. Acquisition and recombination of the “serotype island” resulted in displacement of the native OSA gene cluster and expression of the O12 serotype in the recipients. Serotype switching by recombination has apparently occurred multiple times involving bacteria of various genomic backgrounds. In conclusion, serotype switching in combination with acquisition of an antibiotic resistance determinant most likely contributed to the dissemination of the O12 serotype in clinical settings

Pseudomonas aeruginosa Adaptation to Lungs of Cystic Fibrosis Patients Leads to Lowered Resistance to Phage and Protist Enemies

Pathogenic life styles can lead to highly specialized interactions with host species, potentially resulting in fitness trade-offs in other ecological contexts. Here we studied how adaptation of the environmentally transmitted bacterial pathogen, Pseudomonas aeruginosa, to cystic fibrosis (CF) patients affects its survival in the presence of natural phage (14/1, ΦKZ, PNM and PT7) and protist (Tetrahymena thermophila and Acanthamoebae polyphaga) enemies. We found that most of the bacteria isolated from relatively recently intermittently colonised patients (1-25 months), were innately phage-resistant and highly toxic for protists. In contrast, bacteria isolated from long time chronically infected patients (2-23 years), were less efficient in both resisting phages and killing protists. Moreover, chronic isolates showed reduced killing of wax moth larvae (Galleria mellonella) probably due to weaker in vitro growth and protease expression. These results suggest that P. aeruginosa long-term adaptation to CF-lungs could trade off with its survival in aquatic environmental reservoirs in the presence of microbial enemies, while lowered virulence could reduce pathogen opportunities to infect insect vectors; factors that are both likely to result in poorer environmental transmission. From an applied perspective, phage therapy could be useful against chronic P. aeruginosa lung infections that are often characterized by multidrug resistance: chronic isolates were least resistant to phages and their poor growth will likely slow down the emergence of beneficial resistance mutations

Identification of Metabolic Pathways Essential for Fitness of Salmonella Typhimurium In Vivo

Bacterial infections remain a threat to human and animal health worldwide, and there is an urgent need to find novel targets for intervention. In the current study we used a computer model of the metabolic network of Salmonella enterica serovar Typhimurium and identified pairs of reactions (cut sets) predicted to be required for growth in vivo. We termed such cut sets synthetic auxotrophic pairs. We tested whether these would reveal possible combined targets for new antibiotics by analyzing the performance of selected single and double mutants in systemic mouse infections. One hundred and two cut sets were identified. Sixty-three of these included only pathways encoded by fully annotated genes, and from this sub-set we selected five cut sets involved in amino acid or polyamine biosynthesis. One cut set (asnA/asnB) demonstrated redundancy in vitro and in vivo and showed that asparagine is essential for S. Typhimurium during infection. trpB/trpA as well as single mutants were attenuated for growth in vitro, while only the double mutant was a cut set in vivo, underlining previous observations that tryptophan is essential for successful outcome of infection. speB/speF,speC was not affected in vitro but was attenuated during infection showing that polyamines are essential for virulence apparently in a growth independent manner. The serA/glyA cut-set was found to be growth attenuated as predicted by the model. However, not only the double mutant, but also the glyA mutant, were found to be attenuated for virulence. This adds glycine production or conversion of glycine to THF to the list of essential reactions during infection. One pair (thrC/kbl) showed true redundancy in vitro but not in vivo demonstrating that threonine is available to the bacterium during infection. These data add to the existing knowledge of available nutrients in the intra-host environment, and have identified possible new targets for antibiotics

The Chaperone ClpX Stimulates Expression of Staphylococcus aureus Protein A by Rot Dependent and Independent Pathways

The Clp ATPases (Hsp100) constitute a family of closely related proteins that have protein reactivating and remodelling activities typical of molecular chaperones. In Staphylococcus aureus the ClpX chaperone is essential for virulence and for transcription of spa encoding Protein A. The present study was undertaken to elucidate the mechanism by which ClpX stimulates expression of Protein A. For this purpose, we prepared antibodies directed against Rot, an activator of spa transcription, and demonstrated that cells devoid of ClpX contain three-fold less Rot than wild-type cells. By varying Rot expression from an inducible promoter we showed that expression of Protein A requires a threshold level of Rot. In the absence of ClpX the Rot content is reduced below this threshold level, hence, explaining the substantially reduced Protein A expression in the clpX mutant. Experiments addressed at pinpointing the role of ClpX in Rot synthesis revealed that ClpX is required for translation of Rot. Interestingly, translation of the spa mRNA was, like the rot mRNA, enhanced by ClpX. These data demonstrate that ClpX performs dual roles in regulating Protein A expression, as ClpX stimulates transcription of spa by enhancing translation of Rot, and that ClpX additionally is required for full translation of the spa mRNA. The current findings emphasize that ClpX has a central role in fine-tuning virulence regulation in S. aureus

A Minimal Threshold of c-di-GMP Is Essential for Fruiting Body Formation and Sporulation in Myxococcus xanthus

Generally, the second messenger bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) regulates the switch between motile and sessile lifestyles in bacteria. Here, we show that c-di-GMP is an essential regulator of multicellular development in the social bacterium Myxococcus xanthus. In response to starvation, M. xanthus initiates a developmental program that culminates in formation of spore-filled fruiting bodies. We show that c-di-GMP accumulates at elevated levels during development and that this increase is essential for completion of development whereas excess c-di-GMP does not interfere with development. MXAN3735 (renamed DmxB) is identified as a diguanylate cyclase that only functions during development and is responsible for this increased c-di-GMP accumulation. DmxB synthesis is induced in response to starvation, thereby restricting DmxB activity to development. DmxB is essential for development and functions downstream of the Dif chemosensory system to stimulate exopolysaccharide accumulation by inducing transcription of a subset of the genes encoding proteins involved in exopolysaccharide synthesis. The developmental defects in the dmxB mutant are non-cell autonomous and rescued by co-development with a strain proficient in exopolysaccharide synthesis, suggesting reduced exopolysaccharide accumulation as the causative defect in this mutant. The NtrC-like transcriptional regulator EpsI/Nla24, which is required for exopolysaccharide accumulation, is identified as a c-diGMP receptor, and thus a putative target for DmxB generated c-di-GMP. Because DmxB can be—at least partially—functionally replaced by a heterologous diguanylate cyclase, these results altogether suggest a model in which a minimum threshold level of c-di-GMP is essential for the successful completion of multicellular development in M. xanthus