Deletion of glucose-inhibited division (gidA) gene alters the morphological and replication characteristics of Salmonella enterica Serovar typhimurium.

Salmonella is an important food-borne pathogen that continues to plague the United States food industry. Characterization of bacterial factors involved in food-borne illnesses could help develop new ways to control salmonellosis. We have previously shown that deletion of glucose-inhibited division gene (gidA) significantly altered the virulence potential of Salmonella in both in vitro and in vivo models of infection. Most importantly, the gidA mutant cells displayed a filamentous morphology compared to the wild-type Salmonella cells. In our current study, we investigated the role of GidA in Salmonella cell division using fluorescence and electron microscopy, transcriptional, and proteomic assays. Scanning electron microscopy data indicated a filamentous morphology with few constrictions in the gidA mutant cells. The filamentation of the gidA mutant cells is most likely due to the defect in chromosome segregation, with little to no sign of septa formation observed using fluorescence and transmission electron microscopy. Furthermore, deletion of gidA altered the expression of many genes and proteins responsible for cell division and chromosome segregation as indicated by global transcriptional profiling and semi-quantitative western blot analysis. Taken together, our data indicate GidA as a potential regulator of Salmonella cell division genes.

Fluorescent labeling of cranberry proanthocyanidins with 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein (DTAF).

A novel methodology was developed to elucidate proanthocyanidins (PAC) interaction with extra-intestinal pathogenic Escherichia coli (ExPEC). PAC inhibit ExPEC invasion of epithelial cells and, therefore, may prevent transient gut colonization, conferring protection against subsequent extra-intestinal infections, such as urinary tract infections. Until now PAC have not been chemically labeled with fluorophores. In this work, cranberry PAC were labeled with 5-([4,6-dichlorotriazin-2-yl]amino) fluorescein (DTAF), detected by high-performance liquid chromatography with diode-array detection and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We report single and double fluorescent-labeled PAC with one or two chlorine atoms displaced from DTAF in alkaline pH via nucleophilic substitution. Fluorescent labeling was confirmed by fragmentation experiments using MALDI-TOF/TOF MS. Fluorescent labeled PAC were able to promote ExPEC agglutination when observed with fluorescence microscopy. DTAF tagged PAC may be used to trace the fate of PAC after they agglutinate ExPEC and follow PAC-ExPEC complexes in cell culture assays.

Single-cell elemental analysis of bacteria: quantitative analysis of polyphosphates in Mycobacterium tuberculosis.

More than 1.8 million people die annually from infection with Mycobacterium tuberculosis, the causative agent of tuberculosis. The ability of M. tuberculosis to obtain and distribute micronutrients, including biometals, is known to play a role in its intracellular survival and virulence within a host. Techniques to detect elemental distributions within M. tuberculosis cells have previously been limited to bulk detection methods or low-resolution analyses. Here, we present a method for determining the elemental distribution within M. tuberculosis on a single-cell level, at high (individual nanometer) resolution, using scanning transmission electron microscopy (STEM) in concert with energy-dispersive X-ray spectroscopy (EDS). Results revealed the presence of large polyphosphate granules in all strains of Mycobacteria tested. These persisted even through starvation conditions, and might play a role connected to elemental homeostasis in M. tuberculosis. Associated with the polyphosphate granules were micronutrients such as calcium and magnesium. In addition, we expanded the technique beyond Mycobacteria to show that STEM and EDS could be used as a simple screen to detect the presence or absence of concentrated elements on a single-cell level within all six other bacterial types tested, with minimal processing to the bacteria. Overall, we believe that this technique represents a first step in developing a better understanding of the role that components of the intracellular milieu, including polyphosphates and biometals, play in the pathogenesis of M. tuberculosis, with potential future applications for in vivo analysis.

Life and death in bovine monocytes: the fate of Mycobacterium avium subsp. paratuberculosis.

We previously reported that the number of acid-fast bacilli within Mycobacterium paratuberculosis-infected bovine monocytes increased steadily during an 8-day incubation period in vitro, despite a decrease in the number of viable bacilli as estimated by a radiometric method. In this study, we used differential live/dead staining of bacilli from infected monocytes to show that the percentage of viable bacilli decreased during an 8-day incubation period. We observed poor phagosome-lysosome fusion in monocytes that had ingested viable M. paratuberculosis (30% phagosome-lysosome fusion), while monocytes that ingested heat killed M. paratuberculosis exhibited 94% phagosome-lysosome fusion at 24h after infection. Treatment with the selective Ca(2+)/CaM and PI3 kinase inhibitors (i.e. KN62 and Wortmannin) in combination increased the survival of M. paratuberculosis in bovine monocytes without significantly altering phagosome-lysosome fusion. Scanning electron microscopy suggested that M. paratuberculosis-infected monocytes were less differentiated (smaller and less spreading) than uninfected monocytes at 4 and 8 days of infection. Overall, these data suggest that both multiplication and killing of intracellular M. paratuberculosis occur concomitantly in bovine monocytes. Monocytes in turn may be adversely affected by the bacilli, their products, or factors released from infected monocytes.