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.

In vivo capsular switch in Streptococcus pneumoniae--analysis by whole genome sequencing.

Two multidrug resistant strains of Streptococcus pneumoniae - SV35-T23 (capsular type 23F) and SV36-T3 (capsular type 3) were recovered from the nasopharynx of two adult patients during an outbreak of pneumococcal disease in a New York hospital in 1996. Both strains belonged to the pandemic lineage PMEN1 but they differed strikingly in virulence when tested in the mouse model of IP infection: as few as 1000 CFU of SV36 killed all mice within 24 hours after inoculation while SV35-T23 was avirulent.Whole genome sequencing (WGS) of the two isolates was performed (i) to test if these two isolates belonging to the same clonal type and recovered from an identical epidemiological scenario only differed in their capsular genes? and (ii) to test if the vast difference in virulence between the strains was mostly - or exclusively - due to the type III capsule. WGS demonstrated extensive differences between the two isolates including over 2500 single nucleotide polymorphisms in core genes and also differences in 36 genetic determinants: 25 of which were unique to SV35-T23 and 11 unique to strain SV36-T3. Nineteen of these differences were capsular genes and 9 bacteriocin genes.Using genetic transformation in the laboratory, the capsular region of SV35-T23 was replaced by the type 3 capsular genes from SV36-T3 to generate the recombinant SV35-T3* which was as virulent as the parental strain SV36-T3* in the murine model and the type 3 capsule was the major virulence factor in the chinchilla model as well. On the other hand, a careful comparison of strains SV36-T3 and the laboratory constructed SV35-T3* in the chinchilla model suggested that some additional determinants present in SV36 but not in the laboratory recombinant may also contribute to the progression of middle ear disease. The nature of this determinants remains to be identified.

Preliminary study: treatment with intramuscular interferon beta-1a results in increased levels of IL-12Rß2+ and decreased levels of IL23R+ CD4+ T - Lymphocytes in multiple sclerosis.

BACKGROUND: There are a lack of biomarkers which can be used to predict clinical outcomes for multiple sclerosis (MS) patients receiving interferon beta (IFN-ß). Thus the objective of this study was to characterize changes in CD4+ T-lymphocyte expression in an unbiased manner following initiation of intramuscular (IM) IFN-ß-1a treatment, and then to verify those findings using marker-specific assays. METHODS: Peripheral blood specimens were collected from twenty MS patients before and after treatment with intramuscular (IM) IFN-ß-1a and were used for isolation of mononuclear cells (PBMCs). mRNA expression patterns of negatively-selected CD4+ T-cells from the PBMCs were analyzed using microarray gene expression technology. IL-12 and IL-23 receptor levels on PBMC-derived CD4+ T-cells were analyzed by flow cytometry. The phosphorylation status of Stat4 was measured by performing densitometry on western blots. RESULTS: Microarray analyses demonstrated that mRNA expression of the IL-12Rß2 gene was uniformly up-regulated in response to IFN-ß-1a treatment and was associated with an increased number of IL-12Rß2+ CD4+ T-cells by flow cytometry in 4 of 6 patients. This finding was substantiated by demonstrating that Stat4 phosphorylation, a transcription factor for IL-12, was increased after treatment. Conversely, the number of IL-23R+ CD4+ T-cells was decreased following treatment. CONCLUSIONS: The IL-12 receptor shares a common subunit, the IL-12Rß2, with the IL-23 receptor. Both of these receptors have a probable role in regulating IL-17 and TH-17 cells, important mediators of inflammation in multiple sclerosis (MS). Thus, the changes in the numbers of CD4+ T-cells expressing these receptors in response to IFN-ß-1a treatment may point to an important mechanism of action for this drug, but further large scale studies are needed to confirm these preliminary observations.