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

The PittII Msf-KO strain has decreased survival in macrophages.

<p>Polymyxin B protection assays showing the number of viable bacteria recovered from THP-1 macrophage monolayers 2, 24, 48 and 72 hours after inoculation. Each result represents the mean of 3 wells in 3 biological replicate experiments. <b>(A)</b> Bacterial uptake and survival of: PittII (clinical OM isolate), PittII Msf-KO, the single <i>msfA1</i> gene inserted into the Msf-KO at the <i>ompP1</i> locus: PittII Msf-COMP, and a PittII OMPP1-KO control. Dotted line indicates the limit of detection for the Msf-KO strain. * p<0.05 by one-way weighted ANOVA for independent samples and p<0.05 by Tukey HSD post-hoc test for Msf-KO compared to WT, Msf-COMP and OMPP1-KO. <b>(B)</b> Differentiated THP-1 macrophage monolayers were infected with PittII expressing GFP. At each time-point the cells were washed and fixed. Samples were stained using rabbit anti-NTHi and Alexa Fluor secondary antibodies (red). Red staining indicates extracellular bacteria that are dead. Yellow/orange indicate extracellular bacteria that are viable (expressing GFP). Intracellular bacteria appear green since macrophages were not permeabilized. After 24 hours differential staining of extracellular and intracellular bacteria shows that the majority of the bacteria are inside the macrophages. <b>(C)</b> Bacterial uptake and survival of: NTHi strain 86-028NP (clinical OM isolate) and 86–028 Msf-KO. Dotted line indicates the limit of detection for the Msf-KO strain. * p<0.05 by two-tailed <i>t</i>-test for two independent means. <b>(D)</b> Bacterial uptake and survival of: Rd KW20 (non-encapsulated variant of a type D strain that lacks any Msf gene) and Rd Msf-INS (A mutant with the PittII <i>msfA1</i> gene inserted at the <i>ompP1</i> locus). Dotted line indicates the limit of detection for the Rd KW20 strain. * p<0.05 by two-tailed <i>t</i>-test for two independent means.</p

qRT-PCR confirmation of microarray-based transcriptomic results.

<p>qRT-PCR confirmation of microarray-based transcriptomic results.</p

PittII Msf-KO strain suffers a large competitive disadvantage compared to WT.

<p>Competitive Index (CI) refers to a ratio of mutant bacteria to WT bacteria adjusted for the initial inoculum ratio. CI of 1 indicates no competitive advantage. Data points below 1 indicate a WT advantage and data points above 1 indicate a mutant advantage <b>(A-D)</b>. <b>(A)</b> PittII WT and Msf-KO inoculated 1:1 in planktonic culture for 24 hours (3 experiments with n = 3, error bars denote the standard deviation of each experiment). <b>(B)</b> PittII WT and Msf-KO inoculated 1:1 in biofilm culture harvested over a 6 day period (3 experiments with n = 3, error bars denote the standard error of the mean). CFU were enumerated from both the adherent biofilms as well as the overlying supernatant and the CI was determined from each fraction. *p<0.05 by one-sample two-tailed <i>t</i>-test (μ₀ = 0) on the log CI for both the biofilm and planktonic fractions. <b>(C-E)</b> In two separate experiments six chinchillas were inoculated with 1:1 mixtures of strains and five tissue sites (brain, right and left bullar membranes and right and left bullar effusions) were harvested three days after inoculation. Thus n = 6 for brains and n = 12 for bullar sites. <b>(C)</b> <i>In vivo</i> competition between PittII WT and Msf-KO inoculated 1:1 into 6 animals. Each data point represents a single tissue-site CI value. Points on the X-axis (CI of 0) indicate that no KO bacteria were observed and therefore have an infinitely low CI value. <b>(D)</b> <i>In vivo</i> competition between PittII WT and Msf-COMP inoculated 1:1 into 6 animals compared with the previous data obtained from competition between PittII WT and Msf-KO <i>in vivo</i>. Each data point represents a single tissue-site CI value. Points on the X-axis (CI of 0) indicate that no KO bacteria were observed and thus have an infinitely low CI value. *p<0.05 by two-tailed <i>t</i>-test for two independent means of log cfu data. **p<0.05 by Mann-Whitney U test. n.s. (not significant). <b>(E)</b> Percentage of tissue sites that were positive for bacteria in each of the two <i>in vivo</i> competition experiments (WT vs Msf-KO, and WT vs Msf-COMP). * p<0.05 by two-tailed Fisher-exact test. n.s. (not significant).</p

Genes differentially regulated in the PittII Msf-KO strain compared to the WT strain that are associated with virulence or anaerobiosis.

<p>Genes differentially regulated in the PittII Msf-KO strain compared to the WT strain that are associated with virulence or anaerobiosis.</p

Phylogenetic tree of 210 <i>H</i>. <i>influenzae</i> strains with the distribution of <i>slrV</i> genes and phenotype.

<p>Colored blocks indicate the presence of each type of <i>slrV</i> gene (<i>slrVA-I</i>, see legend). Colored strain names indicate whether the strain is a commensal isolate (blue) or disease isolate (red). Gene data was obtained by whole genome sequencing (24 strains) and by genome hybridization using the custom-designed <i>H</i>. <i>influenzae</i> SGH array (186 strains) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149891#pone.0149891.ref019" target="_blank">19</a>]. Binary data (gene presence or absence) was used to build a distance matrix and the phylogenetic tree was calculated using the neighbor joining method [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149891#pone.0149891.ref114" target="_blank">114</a>]. The interactive Tree of Life web server (<a href="http://itol.embl.de" target="_blank">http://itol.embl.de</a>) was used to visualize the un-rooted tree [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149891#pone.0149891.ref112" target="_blank">112</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149891#pone.0149891.ref113" target="_blank">113</a>].</p

Clinical and geographic origins of 24 whole genome sequenced strains.

<p>Clinical and geographic origins of 24 whole genome sequenced strains.</p

Chromosomal schematic of SlrV locus 1 and architecture of the <i>slrV</i> genes in 24 sequenced <i>H</i>. <i>influenzae</i> strains.

<p>SlrV locus 1 is located between core genes encoding a putative transporting ATPase and peptide chain release factor 1 (<i>prfA</i>). Strain PittII contains four <i>slrVA</i> genes in tandem; two with 2 SLR and two with 4 SLR. These genes correspond to <i>msfA1-4</i> in this manuscript. Only genes predicted to encode full-length products are illustrated. 7 genomes do not contain any full-length <i>slrV</i> gene at this locus. * denotes genomes in which SlrV locus 1 is located on the edges of contig breaks. Therefore it is possible that there are more <i>slrV</i> genes or SLR motifs located in the assembly gaps.</p

Detection of <i>msfA</i> transcripts <i>in vitro</i> and <i>in vivo</i>.

<p>Left panel: RNA was extracted from PittII grown planktonically and as a biofilm in sBHI medium. Right panel: PittII was inoculated bilaterally into the middle ears of three chinchillas via transbullar injection. Animals were euthanized 3h and 24h post-inoculation. Effusions from the middle-ears were harvested immediately and RNA was extracted. Both panels: RNA samples were reverse transcribed (+) or had reverse transcriptase (RT) omitted from the reactions (-). PCR was then performed on + and - RT samples with a primer pair specific to the <i>msfA</i> genes. Due to sequence similarity multiple alleles are amplified. The two different sizes of the four <i>msfA</i> genes make them easily discernible in the gels (white arrows).</p

Phylogenetic inference from SLR genes and protein domains.

<p>Maximum-likelihood trees were calculated using RAxML [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149891#pone.0149891.ref111" target="_blank">111</a>] and visualized with the interactive Tree of Life web server (<a href="http://itol.embl.de" target="_blank">http://itol.embl.de</a>) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149891#pone.0149891.ref112" target="_blank">112</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149891#pone.0149891.ref113" target="_blank">113</a>]. Colored nodes represent the SLR subfamily from which the particular sequence was extracted from (see Legend). <b>(A)</b> Entire SLR gene sequences (n = 79) demonstrating the justification for clustering into different subfamilies. Node labels indicate the strain and strain-specific SGH cluster ID number. <b>(B)</b> 20–26 amino acid long signal peptides (n = 78) from each SLR protein. For the most part the tree structure reflects that of the whole gene tree with individual SLR subfamilies clustering together. Node labels indicate the strain and strain-specific SGH cluster ID number. <b>(C)</b> 22 amino acid C-terminal motif that was identified in 54 SlrV proteins. SlrC proteins do not contain this motif and thus are not included in the analysis. Again, individual SlrV subfamilies cluster together. Node labels indicate the strain and strain-specific SGH cluster ID number. <b>(D)</b> 36 amino acid SLR motifs extracted from the 79 genes containing them (n = 256). Motifs found within the same protein do not cluster together. Node labels indicate the strain, strain-specific SGH cluster ID number, and location of the motif within the CDS (amino acid position). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149891#pone.0149891.s003" target="_blank">S3 Fig</a> presents a rectangular version of this tree without text overlap.</p