Identification and Characterization of msf, a Novel Virulence Factor in Haemophilus influenzae.

Haemophilus influenzae is an opportunistic pathogen. The emergence of virulent, non-typeable strains (NTHi) emphasizes the importance of developing new interventional targets. We screened the NTHi supragenome for genes encoding surface-exposed proteins suggestive of immune evasion, identifying a large family containing Sel1-like repeats (SLRs). Clustering identified ten SLR-containing gene subfamilies, each with various numbers of SLRs per gene. Individual strains also had varying numbers of SLR-containing genes from one or more of the subfamilies. Statistical genetic analyses of gene possession among 210 NTHi strains typed as either disease or carriage found a significant association between possession of the SlrVA subfamily (which we have termed, macrophage survival factor, msf) and the disease isolates. The PittII strain contains four chromosomally contiguous msf genes. Deleting all four of these genes (msfA1-4) (KO) resulted in a highly significant decrease in phagocytosis and survival in macrophages; which was fully complemented by a single copy of the msfA1 gene. Using the chinchilla model of otitis media and invasive disease, the KO strain displayed a significant decrease in fitness compared to the WT in co-infections; and in single infections, the KO lost its ability to invade the brain. The singly complemented strain showed only a partial ability to compete with the WT suggesting gene dosage is important in vivo. The transcriptional profiles of the KO and WT in planktonic growth were compared using the NTHi supragenome array, which revealed highly significant changes in the expression of operons involved in virulence and anaerobiosis. These findings demonstrate that the msfA1-4 genes are virulence factors for phagocytosis, persistence, and trafficking to non-mucosal sites.

Development and validation of an Haemophilus influenzae supragenome hybridization (SGH) array for transcriptomic analyses.

We previously carried out the design and testing of a custom-built Haemophilus influenzae supragenome hybridization (SGH) array that contains probe sequences to 2,890 gene clusters identified by whole genome sequencing of 24 strains of H. influenzae. The array was originally designed as a tool to interrogate the gene content of large numbers of clinical isolates without the need for sequencing, however, the data obtained is quantitative and is thus suitable for transcriptomic analyses. In the current study RNA was extracted from H. influenzae strain CZ4126/02 (which was not included in the design of the array) converted to cDNA, and labelled and hybridized to the SGH arrays to assess the quality and reproducibility of data obtained from these custom-designed chips to serve as a tool for transcriptomics. Three types of experimental replicates were analyzed with all showing very high degrees of correlation, thus validating both the array and the methods used for RNA profiling. A custom filtering pipeline for two-condition unpaired data using five metrics was developed to minimize variability within replicates and to maximize the identification of the most significant true transcriptional differences between two samples. These methods can be extended to transcriptional analysis of other bacterial species utilizing supragenome-based arrays.

Design and validation of a supragenome array for determination of the genomic content of Haemophilus influenzae isolates.

BACKGROUND: Haemophilus influenzae colonizes the human nasopharynx as a commensal, and is etiologically associated with numerous opportunistic infections of the airway; it is also less commonly associated with invasive disease. Clinical isolates of H. influenzae display extensive genomic diversity and plasticity. The development of strategies to successfully prevent, diagnose and treat H. influenzae infections depends on tools to ascertain the gene content of individual isolates. RESULTS: We describe and validate a Haemophilus influenzae supragenome hybridization (SGH) array that can be used to characterize the full genic complement of any strain within the species, as well as strains from several highly related species. The array contains 31,307 probes that collectively cover essentially all alleles of the 2890 gene clusters identified from the whole genome sequencing of 24 clinical H. influenzae strains. The finite supragenome model predicts that these data include greater than 85% of all non-rare genes (where rare genes are defined as those present in less than 10% of sequenced strains). The veracity of the array was tested by comparing the whole genome sequences of eight strains with their hybridization data obtained using the supragenome array. The array predictions were correct and reproducible for ~ 98% of the gene content of all of the sequenced strains. This technology was then applied to an investigation of the gene content of 193 geographically and clinically diverse H. influenzae clinical strains. These strains came from multiple locations from five different continents and Papua New Guinea and include isolates from: the middle ears of persons with otitis media and otorrhea; lung aspirates and sputum samples from pneumonia and COPD patients, blood specimens from patients with sepsis; cerebrospinal fluid from patients with meningitis, as well as from pharyngeal specimens from healthy persons. CONCLUSIONS: These analyses provided the most comprehensive and detailed genomic/phylogenetic look at this species to date, and identified a subset of highly divergent strains that form a separate lineage within the species. This array provides a cost-effective and high-throughput tool to determine the gene content of any H. influenzae isolate or lineage. Furthermore, the method for probe selection can be applied to any species, given a group of available whole genome sequences.