An Oral Inoculation Infant Rabbit Model for Shigella Infection.

Shigella species cause diarrheal disease globally. Shigellosis is typically characterized by bloody stools and colitis with mucosal damage and is the leading bacterial cause of diarrheal death worldwide. After the pathogen is orally ingested, it invades and replicates within the colonic epithelium through mechanisms that rely on its type III secretion system (T3SS). Currently, oral infection-based small animal models to study the pathogenesis of shigellosis are lacking. Here, we found that orogastric inoculation of infant rabbits with Shigella flexneri resulted in diarrhea and colonic pathology resembling that found in human shigellosis. Fasting animals prior to S. flexneri inoculation increased the frequency of disease. The pathogen colonized the colon, where both luminal and intraepithelial foci were observed. The intraepithelial foci likely arise through S. flexneri spreading from cell to cell. Robust S. flexneri intestinal colonization, invasion of the colonic epithelium, and epithelial sloughing all required the T3SS as well as IcsA, a factor required for bacterial spreading and adhesion in vitro Expression of the proinflammatory chemokine interleukin 8 (IL-8), detected with in situ mRNA labeling, was higher in animals infected with wild-type S. flexneri versus mutant strains deficient in icsA or T3SS, suggesting that epithelial invasion promotes expression of this chemokine. Collectively, our findings suggest that oral infection of infant rabbits offers a useful experimental model for studies of the pathogenesis of shigellosis and for testing of new therapeutics.IMPORTANCEShigella species are the leading bacterial cause of diarrheal death globally. The pathogen causes bacillary dysentery, a bloody diarrheal disease characterized by damage to the colonic mucosa and is usually spread through the fecal-oral route. Small animal models of shigellosis that rely on the oral route of infection are lacking. Here, we found that orogastric inoculation of infant rabbits with S. flexneri led to a diarrheal disease and colonic pathology reminiscent of human shigellosis. Diarrhea, intestinal colonization, and pathology in this model were dependent on the S. flexneri type III secretion system and IcsA, canonical Shigella virulence factors. Thus, oral infection of infant rabbits offers a feasible model to study the pathogenesis of shigellosis and to develop and test new therapeutics.

Bacterial spread from cell to cell: beyond actin-based motility.

Several intracellular pathogens display the ability to propagate within host tissues by displaying actin-based motility in the cytosol of infected cells. As motile bacteria reach cell-cell contacts they form plasma membrane protrusions that project into adjacent cells and resolve into vacuoles from which the pathogen escapes, thereby achieving spread from cell to cell. Seminal studies have defined the bacterial and cellular factors that support actin-based motility. By contrast, the mechanisms supporting the formation of protrusions and their resolution into vacuoles have remained elusive. Here, we review recent advances in the field showing that Listeria monocytogenes and Shigella flexneri have evolved pathogen-specific mechanisms of bacterial spread from cell to cell.

The ttpC gene is contained in two of three TonB systems in the human pathogen Vibrio vulnificus, but only one is active in iron transport and virulence.

The TonB system of proteins is required for the energy-dependent active transport of iron-bound substrates across the outer membrane of gram-negative bacteria. We have identified three TonB systems within the human pathogen Vibrio vulnificus. The TonB1 system contains the TonB1, ExbD1, and ExbB1 proteins, whereas both the TtpC2-TonB2 and TtpC3-TonB3 systems contain an additional fourth protein, TtpC. Here we report that TtpC3, although highly related to TtpC2, is inactive in iron transport, whereas TtpC2 is essential for the function of the TtpC2-TonB2 system in V. vulnificus. This protein, together with TonB2, is absolutely required for both the uptake of endogenously produced iron-bound siderophores as well as siderophores produced from other organisms. Through complementation we show that V. vulnificus is capable of using different TtpC2 proteins from other Vibrio species to drive the uptake of multiple siderophores. We have also determined that aerobactin, a common bacterial siderophore involved in virulence of enteric bacteria, can only be brought into the cell using the TtpC2-TonB2 system, indicating an important evolutionary adaptation of TtpC2 and TonB2. Furthermore, in the absence of TonB1, TtpC2 is essential for a fully virulent phenotype as demonstrated using 50% lethal dose (LD(50)) experiments in mice.

Power plays: iron transport and energy transduction in pathogenic vibrios.

The Vibrios are a unique group of bacteria inhabiting a vast array of aquatic environments. Many Vibrio species are capable of infecting a wide assortment of hosts. Some of these species include V. parahaemolyticus, V. alginolyticus, V. vulnificus, V. anguillarum, and V. cholerae. The ability of these organisms to utilize iron is essential in establishing both an infection in their hosts as well as surviving in the environment. Bacteria are able to sequester iron through the secretion of low molecular weight iron chelators termed siderophores. The iron-siderophore complexes are bound by specific outer membrane receptors and are brought through both the outer and inner membranes of the cell. The energy needed to drive this active transport is achieved through the TonB energy transduction system. When first elucidated in E. coli, the TonB system was shown to be a three protein complex consisting of TonB, ExbB and ExbD. Most Vibrio species carry two TonB systems. The second TonB system includes a fourth protein; TtpC, which is essential for TonB2 mediated iron transport. Some Vibrio species have been shown to carry a third TonB system that also includes a TtpC protein.

Molecular and genetic characterization of the TonB2-cluster TtpC protein in pathogenic vibrios.

TtpC is a fourth required protein in the TonB2 energy transduction system in Vibrio anguillarum. TtpC is necessary for iron transport mediated by the TonB2 system and is highly conserved in all pathogenic vibrio species studied to date as well as several marine organisms. We show here that the TtpC proteins from selected pathogenic vibrio species can function with the TonB2 system of V. anguillarum to allow iron transport mediated by a chimeric TonB2 system where the native ExbB2, ExbD2 and TonB2 function with an episomally expressed TtpC in trans from a different species. The discovery that inter-species complementation occurs can be used to identify the functional regions of the TtpC proteins and will lead to an investigation of the mechanism of interaction between the TtpC protein and other members of the TonB2 system.