Long-term evolution of Streptococcus mitis and Streptococcus pneumoniae leads to higher genetic diversity within rather than between human populations.

Evaluation of the apportionment of genetic diversity of human bacterial commensals within and between human populations is an important step in the characterization of their evolutionary potential. Recent studies showed a correlation between the genomic diversity of human commensal strains and that of their host, but the strength of this correlation and of the geographic structure among human populations is a matter of debate. Here, we studied the genomic diversity and evolution of the phylogenetically related oro-nasopharyngeal healthy-carriage Streptococcus mitis and Streptococcus pneumoniae, whose lifestyles range from stricter commensalism to high pathogenic potential. A total of 119 S. mitis genomes showed higher within- and among-host variation than 810 S. pneumoniae genomes in European, East Asian and African populations. Summary statistics of the site-frequency spectrum for synonymous and non-synonymous variation and ABC modelling showed this difference to be due to higher ancestral bacterial population effective size (Ne) in S. mitis, whose genomic variation has been maintained close to mutation-drift equilibrium across (at least many) generations, whereas S. pneumoniae has been expanding from a smaller ancestral bacterial population. Strikingly, both species show limited differentiation among human populations. As genetic differentiation is inversely proportional to the product of effective population size and migration rate (Nem), we argue that large Ne have led to similar differentiation patterns, even if m is very low for S. mitis. We conclude that more diversity within than among human populations and limited population differentiation must be common features of the human microbiome due to large Ne.

Prevalence of phase variable epigenetic invertons among host-associated bacteria.

Type I restriction-modification (R-M) systems consist of a DNA endonuclease (HsdR, HsdM and HsdS subunits) and methyltransferase (HsdM and HsdS subunits). The hsdS sequences flanked by inverted repeats (referred to as epigenetic invertons) in certain Type I R-M systems undergo invertase-catalyzed inversions. Previous studies in Streptococcus pneumoniae have shown that hsdS inversions within clonal populations produce subpopulations with profound differences in the methylome, cellular physiology and virulence. In this study, we bioinformatically identified six major clades of the tyrosine and serine family invertases homologs from 16 bacterial phyla, which potentially catalyze hsdS inversions in the epigenetic invertons. In particular, the epigenetic invertons are highly enriched in host-associated bacteria. We further verified hsdS inversions in the Type I R-M systems of four representative host-associated bacteria and found that each of the resultant hsdS allelic variants specifies methylation of a unique DNA sequence. In addition, transcriptome analysis revealed that hsdS allelic variations in Enterococcus faecalis exert significant impact on gene expression. These findings indicate that epigenetic switches driven by invertases in the epigenetic invertons broadly operate in the host-associated bacteria, which may broadly contribute to bacterial host adaptation and virulence beyond the role of the Type I R-M systems against phage infection.

Selective and non-selective bottlenecks as drivers of the evolution of hypermutable bacterial loci.

Bottlenecks reduce the size of the gene pool within populations of all life forms with implications for their subsequent survival. Here, we examine the effects of bottlenecks on bacterial commensal-pathogens during transmission between, and dissemination within, hosts. By reducing genetic diversity, bottlenecks may alter individual or population-wide adaptive potential. A diverse range of hypermutable mechanisms have evolved in infectious agents that allow for rapid generation of genetic diversity in specific genomic loci as opposed to the variability arising from increased genome-wide mutation rates. These localised hypermutable mechanisms include multi-gene phase variation (PV) of outer membrane components, multi-allele PV of restriction systems and recombination-driven antigenic variation. We review selected experimental and theoretical (mathematical) models pertaining to the hypothesis that localised hypermutation (LH) compensates for fitness losses caused by bottlenecks and discuss whether bottlenecks have driven the evolution of hypermutable loci.

Nasal Pneumococcal Density Is Associated with Microaspiration and Heightened Human Alveolar Macrophage Responsiveness to Bacterial Pathogens.

Rationale: Pneumococcal pneumonia remains a global health problem. Colonization of the nasopharynx with Streptococcus pneumoniae (Spn), although a prerequisite of infection, is the main source of exposure and immunological boosting in children and adults. However, our knowledge of how nasal colonization impacts on the lung cells, especially on the predominant alveolar macrophage (AM) population, is limited.Objectives: Using a controlled human infection model to achieve nasal colonization with 6B serotype, we investigated the effect of Spn colonization on lung cells.Methods: We collected BAL from healthy pneumococcal-challenged participants aged 18-49 years. Confocal microscopy and molecular and classical microbiology were used to investigate microaspiration and pneumococcal presence in the lower airways. AM opsonophagocytic capacity was assessed by functional assays in vitro, whereas flow cytometry and transcriptomic analysis were used to assess further changes on the lung cellular populations.Measurements and Main Results: AMs from Spn-colonized individuals exhibited increased opsonophagocytosis to pneumococcus (11.4% median increase) for approximately 3 months after experimental pneumococcal colonization. AMs also had increased responses against other bacterial pathogens. Pneumococcal DNA detected in the BAL samples of Spn-colonized individuals were positively correlated with nasal pneumococcal density (r = 0.71; P = 0.029). Similarly, AM-heightened opsonophagocytic capacity was correlated with nasopharyngeal pneumococcal density (r = 0.61, P = 0.025).Conclusions: Our findings demonstrate that nasal colonization with pneumococcus and microaspiration prime AMs, leading to brisker responsiveness to both pneumococcus and unrelated bacterial pathogens. The relative abundance of AMs in the alveolar spaces, alongside their potential for nonspecific protection, render them an attractive target for novel vaccines.

Lineage-specific evolution and gene flow in Listeria monocytogenes are independent of bacteriophages.

Listeria monocytogenes is a foodborne pathogen causing systemic infection with high mortality. To allow efficient tracking of outbreaks a clear definition of the genomic signature of a cluster of related isolates is required, but lineage-specific characteristics call for a more detailed understanding of evolution. In our work, we used core genome MLST (cgMLST) to identify new outbreaks combined to core genome SNP analysis to characterize the population structure and gene flow between lineages. Whilst analysing differences between the four lineages of L. monocytogenes we have detected differences in the recombination rate, and interestingly also divergence in the SNP differences between sub-lineages. In addition, the exchange of core genome variation between the lineages exhibited a distinct pattern, with lineage III being the best donor for horizontal gene transfer. Whilst attempting to link bacteriophage-mediated transduction to observed gene transfer, we found an inverse correlation between phage presence in a lineage and the extent of recombination. Irrespective of the profound differences in recombination rates observed between sub-lineages and lineages, we found that the previously proposed cut-off of 10 allelic differences in cgMLST can be still considered valid for the definition of a foodborne outbreak cluster of L. monocytogenes.

Excision-reintegration at a pneumococcal phase-variable restriction-modification locus drives within- and between-strain epigenetic differentiation and inhibits gene acquisition.

Phase-variation of Type I restriction-modification systems can rapidly alter the sequence motifs they target, diversifying both the epigenetic patterns and endonuclease activity within clonally descended populations. Here, we characterize the Streptococcus pneumoniae SpnIV phase-variable Type I RMS, encoded by the translocating variable restriction (tvr) locus, to identify its target motifs, mechanism and regulation of phase variation, and effects on exchange of sequence through transformation. The specificity-determining hsdS genes were shuffled through a recombinase-mediated excision-reintegration mechanism involving circular intermediate molecules, guided by two types of direct repeat. The rate of rearrangements was limited by an attenuator and toxin-antitoxin system homologs that inhibited recombinase gene transcription. Target motifs for both the SpnIV, and multiple Type II, MTases were identified through methylation-sensitive sequencing of a panel of recombinase-null mutants. This demonstrated the species-wide diversity observed at the tvr locus can likely specify nine different methylation patterns. This will reduce sequence exchange in this diverse species, as the native form of the SpnIV RMS was demonstrated to inhibit the acquisition of genomic islands by transformation. Hence the tvr locus can drive variation in genome methylation both within and between strains, and limits the genomic plasticity of S. pneumoniae.

Methylation Warfare: Interaction of Pneumococcal Bacteriophages with Their Host.

Virus-host interactions are regulated by complex coevolutionary dynamics. In Streptococcus pneumoniae, phase-variable type I restriction-modification (R-M) systems are part of the core genome. We hypothesized that the ability of the R-M systems to switch between six target DNA specificities also has a key role in preventing the spread of bacteriophages. Using the streptococcal temperate bacteriophage SpSL1, we show that the variants of both the SpnIII and SpnIV R-M systems are able to restrict invading bacteriophage with an efficiency approximately proportional to the number of target sites in the bacteriophage genome. In addition to restriction of lytic replication, SpnIII also led to abortive infection in the majority of host cells. During lytic infection, transcriptional analysis found evidence of phage-host interaction through the strong upregulation of the nrdR nucleotide biosynthesis regulon. During lysogeny, the phage had less of an effect on host gene regulation. This research demonstrates a novel combined bacteriophage restriction and abortive infection mechanism, highlighting the importance that the phase-variable type I R-M systems have in the multifunctional defense against bacteriophage infection in the respiratory pathogen S. pneumoniaeIMPORTANCE With antimicrobial drug resistance becoming an increasing burden on human health, much attention has been focused on the potential use of bacteriophages and their enzymes as therapeutics. However, the investigations into the physiology of the complex interactions of bacteriophages with their hosts have attracted far less attention, in comparison. This work describes the molecular characterization of the infectious cycle of a bacteriophage in the important human pathogen Streptococcus pneumoniae and explores the intricate relationship between phase-variable host defense mechanisms and the virus. This is the first report showing how a phase-variable type I restriction-modification system is involved in bacteriophage restriction while it also provides an additional level of infection control through abortive infection.

Intracellular survival of Streptococcus pneumoniae in human alveolar macrophages is augmented with HIV infection.

People Living with HIV (PLHIV) are at an increased risk of pneumococcal pneumonia than HIV-uninfected adults, but the reasons for this are still not well understood. We investigated whether alveolar macrophages (AM) mediated control of pneumococcal infection is impaired in PLHIV compared to HIV-uninfected adults. We assessed anti-bactericidal activity against Streptococcus pneumoniae of primary human AM obtained from PLHIV and HIV-uninfected adults. We found that pneumococcus survived intracellularly in AMs at least 24 hours post ex vivo infection, and this was more frequent in PLHIV than HIV-uninfected adults. Corroborating these findings, in vivo evidence showed that PLHIV had a higher propensity for harboring S. pneumoniae within their AMs than HIV-uninfected adults. Moreover, bacterial intracellular survival in AMs was associated with extracellular propagation of pneumococcal infection. Our data suggest that failure of AMs to eliminate S. pneumoniae intracellularly could contribute to the increased risk of pneumococcal pneumonia in PLHIV.

Pathogenic Differences of Type 1 Restriction-Modification Allele Variants in Experimental Listeria monocytogenes Meningitis.

Background:L. monocytogenes meningoencephalitis has a mortality rate of up to 50% and neurofunctional sequelae are common. Type I restriction-modification systems (RMS) are capable of adding methyl groups to the host genome. Some contain multiple sequence recognition (hsdS) genes that recombine, resulting in distinct DNA methylation patterns and patterns of gene expression. These phenotypic switches have been linked to virulence and have recently been discovered in multiple clonal complexes of L. monocytogenes. In the present study, we investigated the significant of RMS on L. monocytogenes virulence during the acute phase of experimental meningitis. Methods:L. monocytogenes strains containing RMS systems were identified, and purified clones enriched for single hsdS alleles were isolated. In vivo, 11-day old Wistar rats were infected with an inoculum containing (a) one of 4 single RMS allele variants (A, B, C, D) treated with amoxicillin (AMX 50 mg/kg/dosis, q8h), (b) a mixture of all 4 variants with or without AMX treatment, or (c) different mixtures of 2 RMS allele variants. At selected time points after infection, clinical and inflammatory parameters, bacterial titers and brain damage were determined. Changes in the relative frequency of the occurring RMS alleles in the inoculum and in CSF or cerebellum of infected animals were analyzed by capillary electrophoresis. Results: We have identified a phase variable RMS locus within L. monocytogenes CC4 and generated stocks that stably expressed each of the possible hsdS genes within that loci. Generation of these allele variants (A, B, C, D) allowed us to determine the methylation pattern associated with each hsdS through SMRT sequencing. In vivo infections with these single allele variants revealed differences in disease severity in that C induced the worst clinical outcome and more pronounced hippocampal apoptosis; D showed the most pronounced weight loss and the highest bacterial titer in the cerebellum. A caused the least severe disease. Conclusion: We identified that L. monocytogenes expressing hsdS (A) causes less damage than when other hsdS genes are expressed. While expression of hsdSC and D worsened the outcome in L. monocytogenes meningitis. We also demonstrate a competitive advantage of variants C and B over variant A in this model. Phenotypical switching may therefore represent a mechanism of virulence regulation during the acute phase of CNS infections with L. monocytogenes.

Deletion of the Zinc Transporter Lipoprotein AdcAII Causes Hyperencapsulation of Streptococcus pneumoniae Associated with Distinct Alleles of the Type I Restriction-Modification System.

The capsule is the dominant Streptococcus pneumoniae virulence factor, yet how variation in capsule thickness is regulated is poorly understood. Here, we describe an unexpected relationship between mutation of adcAII, which encodes a zinc uptake lipoprotein, and capsule thickness. Partial deletion of adcAII in three of five capsular serotypes frequently resulted in a mucoid phenotype that biochemical analysis and electron microscopy of the D39 adcAII mutants confirmed was caused by markedly increased capsule thickness. Compared to D39, the hyperencapsulated ΔadcAII mutant strain was more resistant to complement-mediated neutrophil killing and was hypervirulent in mouse models of invasive infection. Transcriptome analysis of D39 and the ΔadcAII mutant identified major differences in transcription of the Sp_0505-0508 locus, which encodes an SpnD39III (ST5556II) type I restriction-modification system and allelic variation of which correlates with capsule thickness. A PCR assay demonstrated close linkage of the SpnD39IIIC and F alleles with the hyperencapsulated ΔadcAII strains. However, transformation of ΔadcAII with fixed SpnD39III alleles associated with normal capsule thickness did not revert the hyperencapsulated phenotype. Half of hyperencapsulated ΔadcAII strains contained the same single nucleotide polymorphism in the capsule locus gene cps2E, which is required for the initiation of capsule synthesis. These results provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identified an unexpected linkage between capsule thickness and mutation of ΔadcAII Further investigation will be needed to characterize how mutation of adcAII affects SpnD39III (ST5556II) allele dominance and results in the hyperencapsulated phenotype.IMPORTANCE The Streptococcus pneumoniae capsule affects multiple interactions with the host including contributing to colonization and immune evasion. During infection, the capsule thickness varies, but the mechanisms regulating this are poorly understood. We have identified an unsuspected relationship between mutation of adcAII, a gene that encodes a zinc uptake lipoprotein, and capsule thickness. Mutation of adcAII resulted in a striking hyperencapsulated phenotype, increased resistance to complement-mediated neutrophil killing, and increased S. pneumoniae virulence in mouse models of infection. Transcriptome and PCR analysis linked the hyperencapsulated phenotype of the ΔadcAII strain to specific alleles of the SpnD39III (ST5556II) type I restriction-modification system, a system which has previously been shown to affect capsule thickness. Our data provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identify an unexpected link between capsule thickness and ΔadcAII, further investigation of which could further characterize mechanisms of capsule regulation.

Lipopeptidomimetics derived from teixobactin have potent antibacterial activity against Staphylococcus aureus.

A series of lipopeptidomimetics derived from teixobactin have been prepared that probe the role of residues (1-6) as a membrane anchor and the function of enduracididine. The most active compounds, with a farnesyl tail and End10 to Lys10 or Orn10 substitution have potent activity (MIC 8 µg mL-1) against S. aureus. These results pave the way for the synthesis of simple, cost-effective yet potent lipopeptidomimetic antimicrobials.

Phase-variable methylation and epigenetic regulation by type I restriction-modification systems.

Epigenetic modifications in bacteria, such as DNA methylation, have been shown to affect gene regulation, thereby generating cells that are isogenic but with distinctly different phenotypes. Restriction-modification (RM) systems contain prototypic methylases that are responsible for much of bacterial DNA methylation. This review focuses on a distinctive group of type I RM loci that , through phase variation, can modify their methylation target specificity and can thereby switch bacteria between alternative patterns of DNA methylation. Phase variation occurs at the level of the target recognition domains of the hsdS (specificity) gene via reversible recombination processes acting upon multiple hsdS alleles. We describe the global distribution of such loci throughout the prokaryotic kingdom and highlight the differences in loci structure across the various bacterial species. Although RM systems are often considered simply as an evolutionary response to bacteriophages, these multi-hsdS type I systems have also shown the capacity to change bacterial phenotypes. The ability of these RM systems to allow bacteria to reversibly switch between different physiological states, combined with the existence of such loci across many species of medical and industrial importance, highlights the potential of phase-variable DNA methylation to act as a global regulatory mechanism in bacteria.

A random six-phase switch regulates pneumococcal virulence via global epigenetic changes.

Streptococcus pneumoniae (the pneumococcus) is the world's foremost bacterial pathogen in both morbidity and mortality. Switching between phenotypic forms (or 'phases') that favour asymptomatic carriage or invasive disease was first reported in 1933. Here, we show that the underlying mechanism for such phase variation consists of genetic rearrangements in a Type I restriction-modification system (SpnD39III). The rearrangements generate six alternative specificities with distinct methylation patterns, as defined by single-molecule, real-time (SMRT) methylomics. The SpnD39III variants have distinct gene expression profiles. We demonstrate distinct virulence in experimental infection and in vivo selection for switching between SpnD39III variants. SpnD39III is ubiquitous in pneumococci, indicating an essential role in its biology. Future studies must recognize the potential for switching between these heretofore undetectable, differentiated pneumococcal subpopulations in vitro and in vivo. Similar systems exist in other bacterial genera, indicating the potential for broad exploitation of epigenetic gene regulation.

A small RNA encoded in the Rv2660c locus of Mycobacterium tuberculosis is induced during starvation and infection.

Enhanced transcription of the Rv2660c locus in response to starvation of Mycobacterium tuberculosis H37Rv encouraged addition of the predicted Rv2660c protein to an improved vaccine formulation. Using strand-specific RNA sequencing, we show that the up-regulated transcript is in fact a small RNA encoded on the opposite strand to the annotated Rv2660c. The transcript originates within a prophage and is expressed only in strains that carry PhiRv2. The small RNA contains both host and phage sequences and provides a useful biomarker to monitor bacterial starvation during infection and/or non-replicating persistence. Using different approaches we do not find any evidence of Rv2660c at the level of mRNA or protein. Further efforts to understand the mechanism by which Rv2660c improves efficacy of the H56 vaccine are likely to provide insights into the pathology and immunology of tuberculosis.