Streptococcus pneumoniae(pneumococcus), a major human pathogen, is well known for its adaptation to various host environments. Multiple DNA inversions in the three DNA methyltransferasehsdSgenes (hsdSA,hsdSB, andhsdSC) of the colony opacity determinant (cod) locus generate extensive epigenetic and phenotypic diversity. However, it is unclear whether all threehsdSgenes are functional and how the inversions mechanistically occur. In this work, our transcriptional analysis revealed active expression ofhsdSAbut nothsdSBandhsdSC, indicating thathsdSBandhsdSCdo not produce functional proteins and instead act as sources for altering the sequence ofhsdSAby DNA inversions. Consistent with our previous finding that thehsdSinversions are mediated by three pairs of inverted repeats (IR1, IR2, and IR3), this study showed that the 15-bp IR1 and its upstream sequence are strictly required for the inversion betweenhsdSAandhsdSB. Furthermore, a single tyrosine recombinase PsrA catalyzes the inversions mediated by IR1, IR2, and IR3, based on the dramatic loss of these inversions in thepsrAmutant. Surprisingly, PsrA-independent inversions were also detected in thehsdSsequences flanked by the IR2 (298 bp) and IR3 (85 bp) long inverted repeats, which appear to occur spontaneously in the absence of site-specific or RecA-mediated recombination. Because the HsdS subunit is responsible for the sequence specificity of type I restriction modification DNA methyltransferase, these results have revealed thatS. pneumoniaevaries the methylation patterns of the genome DNA (epigenetic status) by employing multiple mechanisms of DNA inversion in thecodlocus.IMPORTANCEStreptococcus pneumoniaeis a major pathogen of human infections with the capacity for adaptation to host environments, but the molecular mechanisms behind this phenomenon remain unclear. Previous studies reveal that pneumococcus extends epigenetic and phenotypic diversity by DNA inversions in three methyltransferasehsdSgenes of thecodlocus. This work revealed that only thehsdSgene that is in the same orientation ashsdMis actively transcribed, but the other two are silent, serving as DNA sources for inversions. While most of thehsdSinversions are catalyzed by PsrA recombinase, the sequences bound by long inverted repeats also undergo inversions via an unknown mechanism. Our results revealed thatS. pneumoniaeswitches the methylation patterns of the genome (epigenetics) by employing multiple mechanisms of DNA inversion.

Certain vaccines are more effective than others against microbial infections, but the molecular mechanisms separating the two types of vaccines are largely undefined. Here, by comparing two vaccines of Streptococcus pneumoniae with identical antigens but different efficacies (pneumococcal conjugate vaccine – PCV13 and pneumococcal polysaccharide vaccine – PPV23), we reveal that superior vaccine protection against blood-borne bacteria is primarily achieved by activating pathogen capture of the sinusoidal endothelial cells (ECs) in the liver. Consistent with its superior protection in humans, PCV13 confers a more potent protection than PPV23 against pneumococcal infection in mice. In vivo real-time imaging and genetic mutagenesis revealed that PCV13 activates both liver ECs and resident macrophages Kupffer cells (KCs) to capture IgG-coated bacteria via IgG Fc gamma receptor (FcγR). In particular, the FcγRIIB-mediated capture by ECs is responsible for PCV13-induced superior protection. In contrast, PPV23 only activates KCs (but not ECs) to achieve a less effective pathogen capture and protection through complement receptor-mediated recognition of IgM- and C3-coated bacteria. These liver-based vaccine protection mechanisms are also found with the vaccines of Neisseria meningitidis and Klebsiella pneumoniae, another two important invasive human pathogens. Our findings have uncovered a novel EC- and FcγRIIB-mediated mechanism in the liver for more efficacious vaccine protection. These findings can serve as in vivo functional readouts to evaluate vaccine efficacy and guide the future vaccine development.One Sentence SummaryVaccine efficacy is defined by FcγRIIB-mediated capture of antibody-coated bacteria via liver sinusoidal endothelial cells.

Site-specific recombination is a DNA breaking and reconstructing process that plays important roles in various cellular pathways for both prokaryotes and eukaryotes. This process requires a site-specific recombinase and direct or inverted repeats. Some tyrosine site-specific recombinases catalyze DNA inversions and regulate subpopulation diversity and phase variation in many bacterial species. In Streptococcus pneumoniae, the PsrA tyrosine recombinase was shown to control DNA inversions in the three DNA methyltransferase hsdS genes of the type I restriction-modification cod locus. Such DNA inversions are mediated by three inverted repeats (IR1, IR2, and IR3). In this work, we purified an untagged form of the PsrA protein and studied its DNA-binding and catalytic features. Gel retardation assays showed that PsrA binds to linear and supercoiled DNAs, containing or not inverted repeats. Nevertheless, DNase I footprinting assays showed that, on linear DNAs, PsrA has a preference for sites that include an IR1 sequence (IR1.1 or IR1.2) and its boundary sequences. Furthermore, on supercoiled DNAs, PsrA was able to generate DNA inversions between specific inverted repeats (IR1, IR2, and IR3), which supports its ability to locate specific target sites. Unlike other site-specific recombinases, PsrA showed reliance on magnesium ions for efficient catalysis of IR1mediated DNA inversions. We discuss that PsrA might find its specific binding sites on the bacterial genome by a mechanism that involves transitory non-specific interactions between protein and DNA.

Streptococcus pneumoniae (pneumococcus) is an important human pathogen that causes pneumonia, meningitis, sepsis, and otitis media. This bacterium normally resides in the nasopharynx as a commensal, but sometimes disseminates to sterile sites of humans and causes local or systemic inflammation. This biphasic behavior of S. pneumoniae is correlated with a reversible switch between the opaque and transparent colony forms on agar plates, a phenomenon referred to as phase variation.The opaque variants appear to be more virulent in animal models of bacteremia but are deficient in nasopharyngeal colonization animal models. In contrast, the transparent variants display higher levels of nasopharyngeal colonization but relatively lower virulence in animal models. We have recently demonstrated that pneumococcal phase variation between these two colony types is caused by a reversible switch of genome DNA methylation (or epigenetic) patterns, which is driven by DNA inversions in the DNA methyltransferase genes. Observation of colony morphology is a simple and useful method to differentiate colonies with different characteristics, such as size, color, and opacity. This protocol describes how to study pneumococcal phase variation in colony morphology with a dissection microscope.