Intracellular Vibrio parahaemolyticus Escapes the Vacuole and Establishes a Replicative Niche in the Cytosol of Epithelial Cells

ABSTRACT Vibrio parahaemolyticus is a globally disseminated Gram-negative marine bacterium and the leading cause of seafood-borne acute gastroenteritis. Pathogenic bacterial isolates encode two type III secretion systems (T3SS), with the second system (T3SS2) considered the main virulence factor in mammalian hosts. For many decades, V. parahaemolyticus has been studied as an exclusively extracellular bacterium. However, the recent characterization of the T3SS2 effector protein VopC has suggested that this pathogen has the ability to invade, survive, and replicate within epithelial cells. Herein, we characterize this intracellular lifestyle in detail. We show that following internalization, V. parahaemolyticus is contained in vacuoles that develop into early endosomes, which subsequently mature into late endosomes. V. parahaemolyticus then escapes into the cytoplasm prior to vacuolar fusion with lysosomes. Vacuolar acidification is an important trigger for this escape. The cytoplasm serves as the pathogen’s primary intracellular replicative niche; cytosolic replication is rapid and robust, with cells often containing over 150 bacteria by the time of cell lysis. These results show how V. parahaemolyticus successfully establishes an intracellular lifestyle that could contribute to its survival and dissemination during infection. IMPORTANCE The marine bacterium V. parahaemolyticus is the leading cause worldwide of seafood-borne acute gastroenteritis. For decades, the pathogen has been studied exclusively as an extracellular bacterium. However, recent results have revealed the pathogen’s ability to invade and replicate within host cells. The present study is the first characterization of the V. parahaemolyticus’ intracellular lifestyle. Upon internalization, V. parahaemolyticus is contained in a vacuole that would in the normal course of events ultimately fuse with a lysosome, degrading the vacuole’s contents. The bacterium subverts this pathway, escaping into the cytoplasm prior to lysosomal fusion. Once in the cytoplasm, it replicates prolifically. Our study provides new insights into the strategies used by this globally disseminated pathogen to survive and proliferate within its host.

Induction of In Vivo Antipolysaccharide Immunoglobulin Responses to Intact Streptococcus pneumoniae Is More Heavily Dependent on Btk-Mediated B-Cell Receptor Signaling than Antiprotein Responses

ABSTRACT The relative role of Btk-dependent B-cell receptor (BCR) signaling in the induction of antipolysaccharide (anti-PS) and antiprotein immunoglobulin (Ig) responses to an intact extracellular bacterium in vivo is unknown. Btklow mice exhibit reduced BCR signaling but largely restore B-cell development. Btklow mice immunized with intact Streptococcus pneumoniae elicit reduced anti-PS but normal antiprotein Ig responses. Immunization of Btklow mice with PS-protein conjugate in saline results in an even more profound defect in the anti-PS but not antiprotein response, which is largely restored by use of a CpG-containing oligodeoxynucleotide as an adjuvant. These data demonstrate a greater dependence on Btk-mediated BCR signaling for physiologic anti-PS relative to antiprotein responses, as well as the existence of a compensatory Toll-like-receptor-mediated signaling pathway naturally triggered in response to intact bacterial pathogens.

Infection-Stimulated Fibrin Deposition Controls Hemorrhage and Limits Hepatic Bacterial Growth during Listeriosis

ABSTRACT Bacterial infections are major causes of human mortality. The activation of coagulation pathways leading to the deposition of insoluble fibrin frequently accompanies bacterial infection, and much attention has focused upon the pathological attributes of infection-stimulated fibrin deposition. Nevertheless, here we present conclusive evidence that infection-stimulated fibrin deposition can perform critical protective functions during bacterial infection. Specifically, we demonstrate that coagulation-impaired fibrin(ogen)-deficient mice, in comparison with genetically matched control mice, display increased mortality upon peritoneal infection with the gram-positive facultative intracellular bacterium Listeria monocytogenes. To distinguish effects of fibrinogen from those of fibrin, we treat wild-type mice with warfarin, an anticoagulant that suppresses fibrin formation without impacting fibrinogen levels. Warfarin treatment exacerbates listeriosis, suggesting that fibrin is the key mediator of protection. With regard to the underlying protective mechanisms, we demonstrate that fibrin(ogen) suppresses anemia, reduces hemorrhagic pathology, and limits bacterial growth during listeriosis. Despite confirming a prior report that fibrin(ogen) promotes the peritoneal clearance of the extracellular bacterium Staphylococcal aureus, we demonstrate that fibrin(ogen) plays little role in controlling peritoneal numbers of L. monocytogenes bacteria or the dissemination of L. monocytogenes bacteria from the peritoneal cavity. Rather, fibrin(ogen) primarily limits the growth of these intracellular bacteria within hepatic tissue. While the pathological potential of excessive infection-stimulated fibrin deposition is well appreciated, our findings reveal that fibrin can function protectively, via multiple mechanisms, during bacterial infection.

Both Innate Immunity and Type 1 Humoral Immunity to Streptococcus pneumoniae Are Mediated by MyD88 but Differ in Their Relative Levels of Dependence on Toll-Like Receptor 2

ABSTRACT Little is known regarding the role of Toll-like receptors (TLRs) in regulating protein- and polysaccharide-specific immunoglobulin (Ig) isotype production in response to an in vivo challenge with an extracellular bacterium. In this report we demonstrate that MyD88−/−, but not TLR2−/−, mice are markedly defective in their induction of multiple splenic proinflammatory cytokine- and chemokine-specific mRNAs after intraperitoneal (i.p.) challenge with heat-killed Streptococcus pneumoniae capsular type 14 (S. pneumoniae type 14). This is correlated with analogous responses in splenic cytokine protein release in vitro following addition of S. pneumoniae type 14. Consistent with these data, naïve MyD88−/−, but not TLR2−/−, mice are more sensitive to killing following i.p. challenge with live S. pneumoniae type 14, relative to responses in wild-type mice. However, prior immunization of MyD88−/− mice with heat-killed S. pneumoniae type 14 protects against an otherwise-lethal challenge with live S. pneumoniae type 14. Surprisingly, both MyD88−/− and TLR2−/− mice exhibit striking and equivalent defects in elicitation of type 1 IgG isotypes (IgG3, IgG2b, and IgG2a), but not the type 2 IgG isotype, IgG1, specific for several protein and polysaccharide antigens, in response to i.p. challenge with heat-killed S. pneumoniae type 14. Of note, the type 1 IgG isotype titers specific for pneumococcal surface protein A are reduced in MyD88−/− mice but not TLR2−/− mice. These data suggest that distinct TLRs may differentially regulate innate versus adaptive humoral immunity to intact S. pneumoniae and are the first to implicate a role for TLR2 in shaping an in vivo type 1 IgG humoral immune response to a gram-positive extracellular bacterium.

The fine structure of the flagellate Mixotricha paradoxa and its associated micro-organisms

Mixotricha is a large polymastigote flagellate from the gut of the termite Mastotermes darwiniensis . Its behaviour and fine structure are described in detail. A study of the method of locomotion, using cinematography, has shown that the four flagella are used not to propel the organism but to steer it; movements are brought about by the co-ordinated undulations of many thousands of spirochaetes which cover most of the body surface. The spirochaetes are attached to small brackets, of complex internal structure, which arise in rows from the cell surface. In addition to one or more spirochaetes, each bracket is also associated with an extracellular bacterium. The fine structures of both spirochaetes and bacteria are described. A network of fibrous strands lies immediately below the cell surface, each of its meshes being associated with one of the over-lying brackets. The cytoplasm contains great numbers of vacuoles, as well as dictyosomes and rough-surfaced membranes. The membranes forming these structures, as well as the membranes of the nuclear envelope and the plasma membrane, are all triple-layered but differ in dimensions and appearance. The rough-surfaced membranes are always associated with intra­-cellular bacteria. The taxonomic position of Mixotricha is discussed in the light of the findings on its structure and behaviour.