As the Lyme disease bacterium Borrelia burgdorferi traverses its enzootic cycle, alternating between a tick vector and a vertebrate host, the spirochete must adapt and persist in the tick midgut under prolonged nutrient stress between blood meals. In this study, we examined the role of the stringent response in tick persistence and in regulation of gene expression during nutrient limitation. Nutritionally starving B. burgdorferi in vitro increased the levels of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), collectively referred to as (p)ppGpp, products of the bifunctional synthetase/hydrolase RelBbu (RelA/SpoT homolog). Conversely, returning B. burgdorferi to a nutrient-rich medium decreased (p)ppGpp levels. B. burgdorferi survival in ticks between the larval and nymph blood meals, and during starvation in vitro, was dependent on RelBbu. Furthermore, normal morphological conversion from a flat-wave shape to a condensed round body (RB) form during starvation was dependent on RelBbu; rel Bbu mutants more frequently formed RBs, but their membranes were compromised. By differential RNA sequencing analyses, we found that RelBbu regulates an extensive transcriptome, both dependent and independent of nutrient stress. The RelBbu regulon includes the glp operon, which is important for glycerol utilization and persistence in the tick, virulence factors and the late phage operon of the 32-kb circular plasmid (cp32) family. In summary, our data suggest that RelBbu globally modulates transcription in response to nutrient stress by increasing (p)ppGpp levels to facilitate B. burgdorferi persistence in the tick.

The spirochetes in the Borrelia burgdorferi sensu lato genospecies group cycle in nature between tick vectors and vertebrate hosts. The current assemblage of B. burgdorferi sensu lato, of which three species cause Lyme disease in humans, originated from a rapid species radiation that occurred near the origin of the clade. All of these species share a unique genome structure that is highly segmented and predominantly composed of linear replicons. One of the circular plasmids is a prophage that exists as several isoforms in each cell and can be transduced to other cells, likely contributing to an otherwise relatively anemic level of horizontal gene transfer, which nevertheless appears to be adequate to permit strong natural selection and adaptation in populations of B. burgdorferi. Although the molecular genetic toolbox is meager, several antibiotic-resistant mutants have been isolated, and the resistance alleles, as well as some exogenous genes, have been fashioned into markers to dissect gene function. Genetic studies have probed the role of the outer membrane lipoprotein OspC, which is maintained in nature by multiple niche polymorphisms and negative frequency-dependent selection. One of the most intriguing genetic systems in B. burgdorferi is vls recombination, which generates antigenic variation during infection of mammalian hosts.

Following invasion of non-phagocytic host cells, Salmonella enterica survives and replicates within a phagosome-like compartment known as the Salmonella -containing vacuole (SCV). It is now well established that SCV biogenesis, like phagosome biogenesis, involves sequential interactions with the endocytic pathway. However, Salmonella is believed to limit these interactions and, in particular, to avoid fusion of terminal lysosomes with the SCV. In this study, we reassessed this process using a high-resolution live-cell imaging approach and found an unanticipated level of interaction between the SCV and the endocytic pathway. Direct interactions, in which late endosomal/lysosomal content was transferred to SCVs, were detected within 30 min of invasion and continued for several hours. Mechanistically, these interactions were very similar to phagosome–lysosome fusion because they were accompanied by rapid acidification of the SCV, could be blocked by chemical perturbation of microtubules or vacuolar acidification and involved the small GTPase Rab7. In comparison with vacuoles containing internalized Escherichia coli or heat-killed Salmonella , SCVs did show some delay of fusion and acidification, although, this appeared to be independent of either type III secretion system. These results provide compelling evidence that inhibition of SCV–lysosome fusion is not the major determinant in establishment of the Salmonella replicative niche in epithelial cells.

Salmonella Typhimurium is a facultative intracellular pathogen that causes acute gastroenteritis in man. Intracellular Salmonella survive and replicate within a modified phagosome known as the Salmonella -containing vacuole (SCV). The onset of intracellular replication is accompanied by the appearance of membrane tubules, called Salmonella -induced filaments (Sifs), extending from the SCV. Sifs are enriched in late endosomal/lysosomal membrane proteins such as lysosome-associated membrane protein 1, but their formation and ability to interact with endosomal compartments are not characterized. In this study, we use live cell imaging techniques to define the dynamics of Sif formation in infected epithelial cells. At early time-points, Sifs are simple tubules extending from the surface of SCVs. These tubules are highly dynamic and exhibit bidirectional, microtubule-dependent movement. At the distal ends of individual Sif tubules, furthest from the SCV, a distinct ‘leader’ domain was often observed. At later times, Sifs develop into highly complex tubular networks that extend throughout the cell and appear less dynamic than nascent Sifs; however, individual tubules continue to display bidirectional dynamics. Sifs can acquire endocytic content by fusion, indicating a sustained interaction with the endocytic pathway. Together, these results show that these Salmonella -induced tubules form a highly dynamic network that involves both microtubule-dependent motility and interactions with endosomal compartments.

Macrophages are an important intracellular niche for Salmonella particularly for systemic infection. The interaction of Salmonella with these cells is mediated by two type III secretion systems (TTSS), encoded on Salmonella pathogenicity islands 1 and 2 (SPI1, SPI2), which mediate distinct phases of the pathogen–host cell interaction. The SPI1 TTSS mediates invasion whereas the SPI2 TTSS is required for intramacrophage survival. Importantly, however, Salmonella can enter macrophages by either SPI1‐dependent invasion or host cell‐mediated phagocytosis. Here, we investigated how the mechanism of internalization affects the intracellular environment and TTSS gene expression. Intracellular bacterial survival depended on the method of entry, because complement‐opsonized and SPI1‐induced Salmonella initiated replication within 8 h whereas immunoglobulin G (IgG)‐opsonized and non‐opsonized Salmonella were initially killed. Analysis of vacuolar pH showed that acidification of the Salmonella‐containing vacuole occurred more rapidly for non‐opsonized or SPI1‐induced Salmonella compared with IgG‐opsonized or complement‐opsonized Salmonella. Finally, quantitative polymerase chain reaction was used to compare the transcriptional profiles of selected SPI1 and SPI2 regulon genes. We found that the magnitude of SPI2 gene induction depended on the mechanism of internalization. Unexpectedly, SPI1 genes, which are rapidly downregulated following SPI1‐mediated invasion, were induced intracellularly following phagocytic uptake. These results reveal another level of complexity in pathogen–macrophage interactions.