Infection of the genitourinary tract with Group B Streptococcus (GBS), an opportunistic gram positive pathogen, is associated with premature rupture of amniotic membrane and preterm birth. In this work, we demonstrate that GBS produces membrane vesicles (MVs) in a serotype independent manner. These MVs are loaded with virulence factors including extracellular matrix degrading proteases and pore forming toxins. Mice chorio-decidual membranes challenged with MVs ex vivo resulted in extensive collagen degradation leading to loss of stiffness and mechanical weakening. MVs when instilled vaginally are capable of anterograde transport in mouse reproductive tract. Intra-amniotic injections of GBS MVs in mice led to upregulation of pro-inflammatory cytokines and inflammation mimicking features of chorio-amnionitis; it also led to apoptosis in the chorio-decidual tissue. Instillation of MVs in the amniotic sac also resulted in intrauterine fetal death and preterm delivery. Our findings suggest that GBS MVs can independently orchestrate events at the feto-maternal interface causing chorio-amnionitis and membrane damage leading to preterm birth or fetal death.

Outcome of host-pathogen encounter is determined by the complex interplay between protective bacterial and host defense strategies. This complexity further amplifies with the existence of cell-to-cell phenotypic heterogeneity in pathogens which remains largely unexplored. In this study, we illustrated that heterogeneous expression of pneumolysin (Ply), a pore-forming toxin of the meningeal pathogen, S. pneumoniae (SPN) gives rise to stochastically different bacterial subpopulations with variable fate during passage across blood-brain barrier (BBB). We demonstrate that Ply mediated damage to pneumococcus containing vacuolar (PCV) membrane leads to recruitment of cytosolic “eat-me” signals, galectin-8 and ubiquitin, targeting SPN for autophagic clearance. However, a majority of high Ply producing subset extensively damages autophagosomes leading to pneumococcal escape into cytosol and efficient clearance by host ubiquitination machinery. Interestingly, a low Ply producing subset halts autophagosomal maturation and evades all intracellular defense mechanisms, promoting its prolonged survival and successful transcytosis across BBB, both in vitro and in vivo. Ply therefore acts as both, sword and shield implying that its smart regulation ensures optimal disease manifestation. Our elucidation of heterogeneity in Ply expression leading to disparate infection outcomes attempts to resolve the dubious role of Ply in pneumococcal pathogenesis.

Collagen is one of the foremost components of tissue extracellular matrix (ECM). It provides strength, elasticity and architecture to the tissue enabling it to bear the wear and tear from external factors like physical stress as well as internal stress factors like inflammation or other pathological conditions. During normal pregnancy or pregnancy related pathological conditions like preterm premature rupture of membranes (PPROM), collagen of the fetal membrane undergoes dynamic remodeling defining biochemical properties of the fetal membrane. The protocol in this article describes the histochemical method to stain total collagen by Picrosirius red stain which is a simple, quick and reliable method. This protocol can be used on paraformaldehyde (PFA) and formaldehyde fixed paraffin embedded tissue sections. We further describe the staining and distribution of collagen in different mouse reproductive tissues and also demonstrate how this technique in combination with polarization microscopy is useful to detect the distribution of different subtypes of collagen.

Meningitis, the inflammation of the protective membrane surrounding the brain and spinal cord (known as meninges), is a condition associated with high mortality rates and permanent neurological sequelae in a significant proportion of survivors. The opportunistic pathogen Streptococcus pneumoniae (SPN/pneumococcus) is the leading cause of bacterial meningitis in adults and older children. Following infection of the lower respiratory tract and subsequent bloodstream invasion, SPN breaches the blood-brain barrier endothelium for invasion of the central nervous system. Transcytosis, a mode of passage through the endothelial cells has been identified as the predominant route of pneumococcal blood-brain barrier trafficking. Herein, we review the interactions enabling SPN invasion into the brain endothelial cells, events involved in the tug-of-war between pneumococcal virulence factors and host intracellular defense machineries and pneumococcal strategies for evasion of host defenses and successful transendothelial trafficking.

The opportunistic pathogen Streptococcus pneumoniae has dual lifestyles: one of an asymptomatic colonizer in the human nasopharynx and the other of a deadly pathogen invading sterile host compartments. The latter triggers an overwhelming inflammatory response, partly driven via pore forming activity of the cholesterol dependent cytolysin (CDC), pneumolysin. Although pneumolysin-induced inflammation drives person-to-person transmission from nasopharynx, the primary reservoir for pneumococcus, it also contributes to high mortality rates, creating a bottleneck that hampers widespread bacterial dissemination, thus acting as a double-edged sword. Serotype 1 ST306, a widespread pneumococcal clone, harbours a non-hemolytic variant of pneumolysin (Ply-NH). Performing crystal structure analysis of Ply-NH, we identified Y150H and T172I as key substitutions responsible for loss of its pore forming activity. We uncovered a novel inter-molecular cation-π interaction, governing formation of the transmembrane β-hairpins (TMH) in the pore state of Ply, which can be extended to other CDCs. H150 in Ply-NH disrupts this interaction, while I172 provides structural rigidity to domain-3, through hydrophobic interactions, inhibiting TMH formation. Loss of pore forming activity enabled improved cellular invasion and autophagy evasion, promoting an atypical intracellular lifestyle for pneumococcus, a finding that was corroborated in in vivo infection models. Attenuation of inflammatory responses and tissue damage promoted tolerance of Ply-NH-expressing pneumococcus in the lower respiratory tract. Adoption of this altered lifestyle may be necessary for ST306 due to its limited nasopharyngeal carriage, with Ply-NH, aided partly by loss of its pore forming ability, facilitating a benign association of SPN in an alternative, intracellular host niche.

25 26 KEY WORDS: Streptococcus pneumoniae, infection tolerance, autophagy, pneumolysin, pore-27 forming toxin, cholesterol dependent cytolysin, cation-π interaction 28 2 ABSTRACT 29 The opportunistic pathogen Streptococcus pneumoniae has dual lifestyles: one of an asymptomatic 30 colonizer in the human nasopharynx and the other of a deadly pathogen invading sterile host 31 compartments. The latter triggers an overwhelming inflammatory response, partly driven via pore 32 forming activity of the cholesterol dependent cytolysin (CDC), pneumolysin. Although 33 pneumolysin-induced inflammation drives person-to-person transmission from nasopharynx, the 34 primary reservoir for pneumococcus, it also contributes to high mortality rates, creating a 35 bottleneck that hampers widespread bacterial dissemination, thus acting as a double-edged sword. 36 Serotype 1 ST306, a widespread pneumococcal clone, harbours a non-hemolytic variant of 37 pneumolysin (Ply-NH). Performing crystal structure analysis of Ply-NH, we identified Y150H and 38 T172I as key substitutions responsible for loss of its pore forming activity. We uncovered a novel 39 inter-molecular cation-π interaction, governing formation of the transmembrane β-hairpins (TMH) 40 in the pore state of Ply, which can be extended to other CDCs. H150 in Ply-NH disrupts this 41 interaction, while I172 provides structural rigidity to domain-3, through hydrophobic interactions, 42 inhibiting TMH formation. Loss of pore forming activity enabled improved cellular invasion and 43 autophagy evasion, promoting an atypical intracellular lifestyle for pneumococcus, a finding that 44 was corroborated in in vivo infection models. Attenuation of inflammatory responses and tissue 45 damage promoted tolerance of Ply-NH-expressing pneumococcus in the lower respiratory tract. 46 Adoption of this altered lifestyle may be necessary for ST306 due to its limited nasopharyngeal 47 157 pneumolysin (Ply-H; 5CR6 and 4QQA) (16, 22). Superposition of Ply-NH structure with the 158 recently reported structures of Ply-H (5CR6 and 4QQA) (16, 22) produced root mean square 159 deviation (r.m.s.d.) of 2.4 and 1.2 Å, respectively, over 471 Cα atoms. Superposition of specific 160 domains, D1-3 of Ply-NH and Ply-H (5CR6) yielded r.m.s.d. of 0.75 Å and alignment of only D4 161

Streptococcus pneumoniae (SPN/pneumococcus), invades myriad of host tissues following efficient breaching of cellular barriers. However, strategies adopted by pneumococcus for evasion of host intracellular defences governing successful transcytosis across host cellular barriers remain elusive. In this study, using brain endothelium as a model host barrier, we observed that pneumococcus containing endocytic vacuoles (PCVs) formed following SPN internalization into brain microvascular endothelial cells (BMECs), undergo early maturation and acidification, with a major subset acquiring lysosome-like characteristics. Exploration of measures that would preserve pneumococcal viability in the lethal acidic pH of these lysosome-like vacuoles revealed a critical role of the two-component system response regulator, CiaR, which has been previously implicated in induction of acid tolerance response. Pyruvate oxidase (SpxB), a key sugar metabolizing enzyme that catalyses oxidative decarboxylation of pyruvate to acetyl phosphate, was found to contribute to acid stress tolerance, presumably via acetyl phosphate-mediated phosphorylation and activation of CiaR, independent of its cognate kinase CiaH. Hydrogen peroxide, the by-product of SpxB catalysed reaction, was also found to improve pneumococcal intracellular survival by oxidative inactivation of lysosomal cysteine cathepsins, thus compromising the degradative capacity of the host lysosomes. Expectedly, a Δ spxB mutant was found to be significantly attenuated in its ability to survive inside the BMEC endocytic vacuoles, reflecting in its reduced transcytosis ability. Collectively, our studies establish SpxB as an important virulence determinant facilitating pneumococcal survival inside host cells, ensuring successful trafficking across host cellular barriers. IMPORTANCE Host cellular barriers have innate immune defences to restrict microbial passage into sterile compartments. Here, by focussing on the blood-brain barrier endothelium, we investigated mechanisms which enable Streptococcus pneumoniae to traverse through host barriers. Pyruvate oxidase, a pneumococcal sugar metabolizing enzyme was found to play a crucial role in this, via generation of acetyl phosphate and hydrogen peroxide. A two-pronged approach consisting of acetyl phosphate-mediated activation of acid tolerance response and hydrogen peroxide-mediated inactivation of lysosomal enzymes enabled pneumococci to maintain viability inside the degradative vacuoles of the brain endothelium, for successful transcytosis across the barrier. Thus, pyruvate oxidase is a key virulence determinant and can potentially serve as a viable candidate for therapeutic interventions for better management of invasive pneumococcal diseases.

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