Stefan P. W. de Vries

Genome analysis of Moraxella catarrhalis strain RH4, a human respiratory tract pathogen

Moraxella catarrhalis is an emerging human-restricted respiratory tract pathogen that is a common cause of childhood otitis media and exacerbations of chronic obstructive pulmonary disease in adults. Here, we report the first completely assembled and annotated genome sequence of an isolate of M. catarrhalis, strain RH4, which originally was isolated from blood of an infected patient. The RH4 genome consists of 1,863,286 nucleotides that form 1,886 protein-encoding genes. Comparison of the RH4 genome to the ATCC 43617 contigs demonstrated that the gene content of both strains is highly conserved. In silico phylogenetic analyses based on both 16S rRNA and multilocus sequence typing revealed that RH4 belongs to the seroresistant lineage. We were able to identify almost the entire repertoire of known M. catarrhalis virulence factors and mapped the members of the biosynthetic pathways for lipooligosaccharide, peptidoglycan, and type IV pili. Reconstruction of the central metabolic pathways suggested that RH4 relies on fatty acid and acetate metabolism, as the genes encoding the enzymes required for the glyoxylate pathway, the tricarboxylic acid cycle, the gluconeogenic pathway, the nonoxidative branch of the pentose phosphate pathway, the beta-oxidation pathway of fatty acids, and acetate metabolism were present. Moreover, pathways important for survival under challenging in vivo conditions, such as the iron-acquisition pathways, nitrogen metabolism, and oxidative stress responses, were identified. Finally, we showed by microarray expression profiling that approximately 88% of the predicted coding sequences are transcribed under in vitro conditions. Overall, these results provide a foundation for future research into the mechanisms of M. catarrhalis pathogenesis and vaccine development.

Multicomponent Moraxella catarrhalis outer membrane vesicles induce an inflammatory response and are internalized by human epithelial cells.

Moraxella catarrhalis is an emerging human respiratory pathogen in patients with chronic obstructive pulmonary disease (COPD) and in children with acute otitis media. The specific secretion machinery known as outer membrane vesicles (OMVs) is a mechanism by which Gram‐negative pathogens interact with host cells during infection. We identified 57 proteins in M. catarrhalis OMVs using a proteomics approach combining two‐dimensional SDS‐PAGE and MALDI‐TOF mass spectrometry analysis. The OMVs contained known surface proteins such as ubiquitous surface proteins (Usp) A1/A2, and Moraxella IgD‐binding protein (MID). Most of the proteins are adhesins/virulence factors triggering the immune response, but also aid bacteria to evade the host defence. FITC‐stained OMVs bound to lipid raft domains in alveolar epithelial cells and were internalized after interaction with Toll‐like receptor 2 (TLR2), suggesting a delivery to the host tissue of a large and complex group of OMV‐attributed proteins. Interestingly, OMVs modulated the pro‐inflammatory response in epithelial cells, and UspA1‐bearing OMVs were found to specifically downregulate the reaction. When mice were exposed to OMVs, a pulmonary inflammation was clearly seen. Our findings indicate that Moraxella OMVs are highly biologically active, transport main bacterial virulence factors and may modulate the epithelial pro‐inflammatory response.

Molecular Aspects of Moraxella catarrhalis Pathogenesis

SUMMARY In recent years, Moraxella catarrhalis has established its position as an important human mucosal pathogen, no longer being regarded as just a commensal bacterium. Further, current research in the field has led to a better understanding of the molecular mechanisms involved in M. catarrhalis pathogenesis, including mechanisms associated with cellular adherence, target cell invasion, modulation of the hosts immune response, and metabolism. Additionally, in order to be successful in the host, M. catarrhalis has to be able to interact and compete with the commensal flora and overcome stressful environmental conditions, such as nutrient limitation. In this review, we provide a timely overview of the current understanding of the molecular mechanisms associated with M. catarrhalis virulence and pathogenesis.

Comparative analysis and supragenome modeling of twelve Moraxella catarrhalis clinical isolates

BackgroundM. catarrhalis is a gram-negative, gamma-proteobacterium and an opportunistic human pathogen associated with otitis media (OM) and exacerbations of chronic obstructive pulmonary disease (COPD). With direct and indirect costs for treating these conditions annually exceeding

Genome-wide association of functional traits linked with Campylobacter jejuni survival from farm to fork

33 billion in the United States alone, and nearly ubiquitous resistance to beta-lactam antibiotics among M. catarrhalis clinical isolates, a greater understanding of this pathogens genome and its variability among isolates is needed.ResultsThe genomic sequences of ten geographically and phenotypically diverse clinical isolates of M. catarrhalis were determined and analyzed together with two publicly available genomes. These twelve genomes were subjected to detailed comparative and predictive analyses aimed at characterizing the supragenome and understanding the metabolic and pathogenic potential of this species. A total of 2383 gene clusters were identified, of which 1755 are core with the remaining 628 clusters unevenly distributed among the twelve isolates. These findings are consistent with the distributed genome hypothesis (DGH), which posits that the species genome possesses a far greater number of genes than any single isolate. Multiple and pair-wise whole genome alignments highlight limited chromosomal re-arrangement.ConclusionsM. catarrhalis gene content and chromosomal organization data, although supportive of the DGH, show modest overall genic diversity. These findings are in stark contrast with the reported heterogeneity of the species as a whole, as wells as to other bacterial pathogens mediating OM and COPD, providing important insight into M. catarrhalis pathogenesis that will aid in the development of novel therapeutic regimens.

From microbial gene essentiality to novel antimicrobial drug targets

Campylobacter jejuni is a major cause of bacterial gastroenteritis worldwide, primarily associated with the consumption of contaminated poultry. C. jejuni lineages vary in host range and prevalence in human infection, suggesting differences in survival throughout the poultry processing chain. From 7343 MLST-characterised isolates, we sequenced 600 C. jejuni and C. coli isolates from various stages of poultry processing and clinical cases. A genome-wide association study (GWAS) in C. jejuni ST-21 and ST-45 complexes identified genetic elements over-represented in clinical isolates that increased in frequency throughout the poultry processing chain. Disease-associated SNPs were distinct in these complexes, sometimes organised in haplotype blocks. The function of genes containing associated elements was investigated, demonstrating roles for cj1377c in formate metabolism, nuoK in aerobic survival and oxidative respiration, and cj1368-70 in nucleotide salvage. This work demonstrates the utility of GWAS for investigating transmission in natural zoonotic pathogen populations and provides evidence that major C. jejuni lineages have distinct genotypes associated with survival, within the host specific niche, from farm to fork.

Characterization of the molecular interplay between Moraxella catarrhalis and human respiratory tract epithelial cells

BackgroundBacterial respiratory tract infections, mainly caused by Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are among the leading causes of global mortality and morbidity. Increased resistance of these pathogens to existing antibiotics necessitates the search for novel targets to develop potent antimicrobials.ResultHere, we report a proof of concept study for the reliable identification of potential drug targets in these human respiratory pathogens by combining high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics. Approximately 20% of all genes in these three species were essential for growth and viability, including 128 essential and conserved genes, part of 47 metabolic pathways. By comparing these essential genes to the human genome, and a database of genes from commensal human gut microbiota, we identified and excluded potential drug targets in respiratory tract pathogens that will have off-target effects in the host, or disrupt the natural host microbiota. We propose 249 potential drug targets, 67 of which are targets for 75 FDA-approved antimicrobials and 35 other researched small molecule inhibitors. Two out of four selected novel targets were experimentally validated, proofing the concept.ConclusionHere we have pioneered an attempt in systematically combining the power of high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics to discover potential drug targets at genome-scale. By circumventing the time-consuming and expensive laboratory screens traditionally used to select potential drug targets, our approach provides an attractive alternative that could accelerate the much needed discovery of novel antimicrobials.

Changes in duodenal tissue-associated microbiota following hookworm infection and consecutive gluten challenges in humans with coeliac disease

Moraxella catarrhalis is a mucosal pathogen that causes childhood otitis media and exacerbations of chronic obstructive pulmonary disease in adults. During the course of infection, M. catarrhalis needs to adhere to epithelial cells of different host niches such as the nasopharynx and lungs, and consequently, efficient adhesion to epithelial cells is considered an important virulence trait of M. catarrhalis. By using Tn-seq, a genome-wide negative selection screenings technology, we identified 15 genes potentially required for adherence of M. catarrhalis BBH18 to pharyngeal epithelial Detroit 562 and lung epithelial A549 cells. Validation with directed deletion mutants confirmed the importance of aroA (3-phosphoshikimate 1-carboxyvinyl-transferase), ecnAB (entericidin EcnAB), lgt1 (glucosyltransferase), and MCR_1483 (outer membrane lipoprotein) for cellular adherence, with ΔMCR_1483 being most severely attenuated in adherence to both cell lines. Expression profiling of M. catarrhalis BBH18 during adherence to Detroit 562 cells showed increased expression of 34 genes in cell-attached versus planktonic bacteria, among which ABC transporters for molybdate and sulfate, while reduced expression of 16 genes was observed. Notably, neither the newly identified genes affecting adhesion nor known adhesion genes were differentially expressed during adhesion, but appeared to be constitutively expressed at a high level. Profiling of the transcriptional response of Detroit 562 cells upon adherence of M. catarrhalis BBH18 showed induction of a panel of pro-inflammatory genes as well as genes involved in the prevention of damage of the epithelial barrier. In conclusion, this study provides new insight into the molecular interplay between M. catarrhalis and host epithelial cells during the process of adherence.

Genetic requirements for Moraxella catarrhalis growth under iron-limiting conditions

A reduced diversity of the gastrointestinal commensal microbiota is associated with the development of several inflammatory diseases. Recent reports in humans and animal models have demonstrated the beneficial therapeutic effects of infections by parasitic worms (helminths) in some inflammatory disorders, such as inflammatory bowel disease (IBD) and coeliac disease (CeD). Interestingly, these studies have described how helminths may alter the intestinal microbiota, potentially representing a mechanism by which they regulate inflammation. However, for practical reasons, these reports have primarily analysed the faecal microbiota. In the present investigation, we have assessed, for the first time, the changes in the microbiota at the site of infection by a parasitic helminth (hookworm) and gluten-dependent inflammation in humans with CeD using biopsy tissue from the duodenum. Hookworm infection and gluten exposure were associated with an increased abundance of species within the Bacteroides phylum, as well as increases in the richness and diversity of the tissue-resident microbiota within the intestine, results that are consistent with previous reports using other helminth species in humans and animal models. Hence, this may represent a mechanism by which parasitic helminths may restore intestinal immune homeostasis and exert a therapeutic benefit in CeD, and potentially other inflammatory disorders.

Genome sequence of Moraxella catarrhalis RH4, an isolate of seroresistant lineage.

Iron sequestration by the human host is a first line defence against respiratory pathogens like Moraxella catarrhalis, which consequently experiences a period of iron starvation during colonization. We determined the genetic requirements for M. catarrhalis BBH18 growth during iron starvation using the high‐throughput genome‐wide screening technology genomic array footprinting (GAF). By subjecting a large random transposon mutant library to growth under iron‐limiting conditions, mutants of the MCR_0996‐rhlB‐yggW operon, rnd, and MCR_0457 were negatively selected. Growth experiments using directed mutants confirmed the GAF phenotypes with ΔyggW (putative haem‐shuttling protein) and ΔMCR_0457 (hypothetical protein) most severely attenuated during iron starvation, phenotypes which were restored upon genetic complementation of the deleted genes. Deletion of yggW resulted in similar attenuated phenotypes in three additional strains. Transcriptional profiles of ΔyggW and ΔMCR_0457 were highly altered with 393 and 192 differentially expressed genes respectively. In all five mutants, expression of nitrate reductase genes was increased and of nitrite reductase decreased, suggesting an impaired aerobic respiration. Alteration of iron metabolism may affect nasopharyngeal colonization as adherence of all mutants to respiratory tract epithelial cells was attenuated. In conclusion, we elucidated the genetic requirements for M. catarrhalis growth during iron starvation and characterized the roles of the identified genes in bacterial growth and host interaction.