Strains of bacterial species induce a greatly varied acute adaptive immune response: The contribution of the accessory genome

<div><p>A fundamental question in human susceptibility to bacterial infections is to what extent variability is a function of differences in the pathogen species or in individual humans. To focus on the pathogen species, we compared in the same individual the human adaptive T and B cell immune response to multiple strains of two major human pathogens, <i>Staphylococcus aureus</i> and <i>Streptococcus pyogenes</i>. We found wide variability in the acute adaptive immune response induced by various strains of a species, with a unique combination of activation within the two arms of the adaptive response. Further, this was also accompanied by a dramatic difference in the intensity of the specific protective T helper (Th) response. Importantly, the same immune response differences induced by the individual strains were maintained across multiple healthy human donors. A comparison of isogenic phage KO strains, demonstrated that of the pangenome, prophages were the major contributor to inter-strain immune heterogeneity, as the T cell response to the remaining “core genome” was noticeably blunted. Therefore, these findings extend and modify the notion of an adaptive response to a pathogenic bacterium, by implying that the adaptive immune response signature of a bacterial species should be defined either per strain or alternatively to the species’ ‘core genome’, common to all of its strains. Further, our results demonstrate that the acquired immune response variation is as wide among different strains within a single pathogenic species as it is among different humans, and therefore may explain in part the clinical heterogeneity observed in patients infected with the same species.</p></div

The inter-individual variability of the immune response to a strain vs the intra-individual variability of the immune response to various strains within a species.

<p><b>A, B, C, and D</b>. The percentage Coefficient of Variation (CV) in % CD3+CD4+ proliferation (A), %IFNγ expressing CD4+ cells (B), % B cell Proliferation (C), and % IgG expression by proliferating B cells (D), was calculated across bacterial strains vs donors. Expressed is the mean percentage CV±2*SEM.</p

The effect of phage KO on adaptive immune response.

<p><b>A. and C</b>. CFSE labeled PBMC from 10 donors were stimulated with strains Newman or TB4 (Newman KO of its 4 phages) as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006726#ppat.1006726.g001" target="_blank">Fig 1A</a>, stained and analyzed by FACS for % proliferation (CFSE dilution) of live CD3+CD4+ cells (A left), % IFNγ expression by proliferating live CD3+CD4+ cells (A right), % proliferation of B cell (live CD3-CD19+, C left), or % IgG expression by live B cells (C right). <b>B</b>. Example of FACS plot of one of the donors in Figure A. <b>D</b>. Intracellular staining for IFNγ, IL17A expression in proliferating live CD3+CD4+ cells following stimulation with either Newman or TB4. <b>E</b>. As in D, but staining for transcription factor Tbet and RORγt. <b>F. and G</b>. are as in A and C left, respectively, but after stimulation with wild type <i>Streptococcus pyogenes</i> M1 strain SF370 or its complete phage KO. <b>H</b>. Intracellular staining for IFNγ, IL17A expression in proliferating live CD3+CD4+ cells following stimulation with either wild type <i>Streptococcus pyogenes</i> M1 strain SF370 or its complete phage KO (CEM1ΔΦ). <b>I</b>. The MANOVA test for difference between bivariate means of % T cell proliferation and % B cell proliferation cells following stimulation with either TB4 or <i>Streptococcus pyogenes</i> M1 (SF370) complete phage KO (CEM1ΔΦ) strains.</p

The Phage Lysin PlySs2 Decolonizes <i>Streptococcus suis</i> from Murine Intranasal Mucosa

<div><p><i>Streptococcus suis</i> infects pigs worldwide and may be zoonotically transmitted to humans with a mortality rate of up to 20%. <i>S</i>. <i>suis</i> has been shown to develop <i>in vitro</i> resistance to the two leading drugs of choice, penicillin and gentamicin. Because of this, we have pursued an alternative therapy to treat these pathogens using bacteriophage lysins. The bacteriophage lysin PlySs2 is derived from an <i>S</i>. <i>suis</i> phage and displays potent lytic activity against most strains of that species including serotypes 2 and 9. At 64 μg/ml, PlySs2 reduced multiple serotypes of <i>S</i>. <i>suis</i> by 5 to 6-logs within 1 hour <i>in vitro</i> and exhibited a minimum inhibitory concentration (MIC) of 32 μg/ml for a <i>S</i>. <i>suis</i> serotype 2 strain and 64 μg/ml for a serotype 9 strain. Using a single 0.1-mg dose, the colonizing <i>S</i>. <i>suis</i> serotype 9 strain was reduced from the murine intranasal mucosa by >4 logs; a 0.1-mg dose of gentamicin reduced <i>S</i>. <i>suis</i> by <3-logs. A combination of 0.05 mg PlySs2 + 0.05 mg gentamicin reduced <i>S</i>. <i>suis</i> by >5-logs. While resistance to gentamicin was induced after systematically increasing levels of gentamicin in an <i>S</i>. <i>suis</i> culture, the same protocol resulted in no observable resistance to PlySs2. Thus, PlySs2 has both broad and high killing activity against multiple serotypes and strains of <i>S</i>. <i>suis</i>, making it a possible tool in the control and prevention of <i>S</i>. <i>suis</i> infections in pigs and humans.</p></div

PlySs2 was bactericidal to nearly all strains of <i>S</i>. <i>suis</i>.

<p>Bacteria were grown to log-phase. After exposure to 64 μg/ml PlySs2 in buffer A for 60 min in 96-well plates, bacteria were serially diluted and plated to BHI agar for CFU enumeration. The CFU numbers of most <i>S</i>. <i>suis</i> strains dropped by 5 to 6 logs after PlySs2 treatment including the type strain S735 and the pathogenic strains 10 and 7997. Death (log fold kill) was calculated as -log[(CFUs in the test condition) ÷ (CFUs in the control condition)].</p

PlySs2 displayed activity against almost all strains of <i>S</i>. <i>suis</i>.

<p>Bacteria in logarithmic growth were exposed to 32 μg/ml PlySs2 for 30 minutes in PB (for 60-minute readings, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169180#pone.0169180.s004" target="_blank">S3 Fig</a>). The activity was measured by OD₆₀₀ reduction. To normalize and combine values from multiple tests, the final OD₆₀₀ of the treated samples was divided by the final OD₆₀₀ of the untreated samples. An OD₆₀₀ ratio of 1.0 indicates no lysis, while an OD₆₀₀ ratio of ~0.02 indicates complete lysis.</p

Location of the major bacteriophage elements in the chromosome of SF370.

<p>Triangles represent the location of the four main bacteriophage-like elements in the circular genome of <i>S</i>. <i>pyogenes</i> strain SF370. Numbers in triangles represent the corresponding phage elements with their size and encoded virulence factors identified in the table below. Derived from GenBank nucleotide accession number: AE004092.</p