Cytosolic replication in epithelial cells fuels intestinal expansion and chronic fecal shedding of Salmonella Typhimurium.

Persistent and intermittent fecal shedding, hallmarks of Salmonella infections, are important for fecal-oral transmission. In the intestine, Salmonella enterica serovar Typhimurium (STm) actively invades intestinal epithelial cells (IECs) and survives in the Salmonella-containing vacuole (SCV) and the cell cytosol. Cytosolic STm replicate rapidly, express invasion factors, and induce extrusion of infected epithelial cells into the intestinal lumen. Here, we engineered STm that self-destruct in the cytosol (STmCytoKill), but replicates normally in the SCV, to examine the role of cytosolic STm in infection. Intestinal expansion and fecal shedding of STmCytoKill are impaired in mouse models of infection. We propose a model whereby repeated rounds of invasion, cytosolic replication, and release of invasive STm from extruded IECs fuels the high luminal density required for fecal shedding.

Bacterial protein domains with a novel Ig-like fold target human CEACAM receptors.

Streptococcus agalactiae, also known as group B Streptococcus (GBS), is the major cause of neonatal sepsis in humans. A critical step to infection is adhesion of bacteria to epithelial surfaces. GBS adhesins have been identified to bind extracellular matrix components and cellular receptors. However, several putative adhesins have no host binding partner characterised. We report here that surface-expressed ß protein of GBS binds to human CEACAM1 and CEACAM5 receptors. A crystal structure of the complex showed that an IgSF domain in ß represents a novel Ig-fold subtype called IgI3, in which unique features allow binding to CEACAM1. Bioinformatic assessment revealed that this newly identified IgI3 fold is not exclusively present in GBS but is predicted to be present in adhesins from other clinically important human pathogens. In agreement with this prediction, we found that CEACAM1 binds to an IgI3 domain found in an adhesin from a different streptococcal species. Overall, our results indicate that the IgI3 fold could provide a broadly applied mechanism for bacteria to target CEACAMs.

Foodborne infection of mice with Salmonella Typhimurium.

The bacterial pathogen Salmonella enterica serovar Typhimurium is one of the most common causes of foodborne disease in humans and is also an important model system for bacterial pathogenesis. Oral inoculation of C57Bl/6 mice, which are genetically susceptible to Salmonella, results in systemic infection but the murine intestine is not efficiently colonized unless the intestinal microbiota is disrupted. Pretreatment of C57Bl/6 mice with streptomycin, followed by oral inoculation with Salmonella Typhimurium results in colitis resembling human intestinal Salmonellosis. The predominant method of delivery of bacteria is oral gavage, during which organisms are deposited directly into the stomach via a feeding needle. Although convenient, this method can be stressful for mice, and may lead to unwanted tracheal or systemic introduction of bacteria. Here, we developed a method for oral infection of mice by voluntary consumption of regular mouse chow inoculated with bacteria. Mice readily ate chow fragments containing up to 108 CFU Salmonella, allowing for a wide range of infectious doses. In mice pretreated with streptomycin, infection with inoculated chow resulted in reproducible infections with doses as low as 103 CFU. Mice not treated with streptomycin, as well as resistant Nramp1 reconstituted C57Bl/6J mice, were also readily infected using this method. In summary, voluntary consumption of chow inoculated with Salmonella represents a natural route of infection for foodborne salmonellosis and a viable alternative to oral gavage.

Dissecting Flavivirus Biology in Salivary Gland Cultures from Fed and Unfed Ixodes scapularis (Black-Legged Tick).

The Ixodes scapularis tick transmits a number of pathogens, including tick-borne flaviviruses (TBFVs). In the United States, confirmed human infections with the Powassan virus (POWV) TBFV have a fatality rate of ∼10% and are increasing in incidence. Tick salivary glands (SGs) serve as an organ barrier to TBFV transmission, and little is known regarding the location of TBFV infection in SGs from fed ticks. Previous studies showed I. scapularis vanin (VNN) involved with TBFV infection of I. scapularis ISE6 embryonic cells, suggesting a potential role for this gene. The overall goal of this study was to use SG cultures to compare data on TBFV biology in SGs from fully engorged, replete (fed) ticks and from unfed ticks. TBFV multiplication was higher in SGs from fed ticks than in those from unfed ticks. Virus-like particles were observed only in granular acini of SGs from unfed ticks. The location of TBFV infection of SGs from fed ticks was observed in cells lining lobular ducts and trachea but not observed in acini. Transcript knockdown of VNN decreased POWV multiplication in infected SG cultures from both fed and unfed ticks. This work was the first to identify localization of TBFV multiplication in SG cultures from a fed tick and a tick transcript important for POWV multiplication in the tick SG, an organ critical for TBFV transmission. This research exemplifies the use of SG cultures in deciphering TBFV biology in the tick and as a translational tool for screening and identifying potential tick genes as potential countermeasure targets.IMPORTANCE Tick-borne flaviviruses (TBFVs) are responsible for more than 15,000 human disease cases each year, and Powassan virus lineage 2 (POWV-L2) deer tick virus has been a reemerging threat in North America over the past 20 years. Rapid transmission of TBFVs in particular emphasizes the importance of preventing tick bites, the difficulty in developing countermeasures to prevent transmission, and the importance of understanding TBFV infection in tick salivary glands (SGs). Tick blood feeding is responsible for phenomenal physiological changes and is associated with changes in TBFV multiplication within the tick and in SGs. Using SG cultures from Ixodes scapularis female ticks, the primary aims of this study were to identify cellular localization of virus-like particles in acini of infected SGs from fed and unfed ticks, localization of TBFV infection in infected SGs from fed ticks, and a tick transcript (with associated metabolic function) involved in POWV-L2 infection in SG cultures.