Determination of Growth Rate and Virulence Plasmid Copy Number During Yersinia pseudotuberculosis Infection Using Droplet Digital PCR.

Pathogenic bacteria have evolved the ability to evade their host defenses and cause diseases. Virulence factors encompass a wide range of adaptations that allow pathogens to survive and proliferate in the hostile host environment during successful infection. In human pathogenic Yersinia species, the potent type III secretion system (T3SS) and other essential virulence factors are encoded on a virulence plasmid. Here, we investigated the bacterial growth rate and plasmid copy number following a Yersinia infection using droplet digital PCR (ddPCR). ddPCR is an exceptionally sensitive, highly precise, and cost-efficient method. It enables precise quantification even from very small amounts of target DNA. This method also enables analysis of complex samples with large amounts of interfering DNA, such as infected tissues or microbiome studies.

Calcium-responsive plasmid copy number regulation is dependent on discrete YopD domains in Yersinia pseudotuberculosis.

Yersinia pathogenicity depends mainly on a Type III Secretion System (T3SS) responsible for translocating effector proteins into the eukaryotic target cell cytosol. The T3SS is encoded on a 70 kb, low copy number virulence plasmid, pYV. A key T3SS regulator, YopD, is a multifunctional protein and consists of discrete modular domains that are essential for pore formation and translocation of Yop effectors. In Y. pseudotuberculosis, the temperature-dependent plasmid copy number increase that is essential for elevated T3SS gene dosage and virulence is also affected by YopD. Here, we found that the presence of intracellular YopD results in increased levels of the CopA-RNA and CopB, two inhibitors of plasmid replication. Secretion of YopD leads to decreased expression of copA and copB, resulting in increased plasmid copy number. Moreover, using a systematic mutagenesis of YopD mutants, we demonstrated that the same discrete modular domains important for YopD translocation are also necessary for both the regulation of plasmid copy number as well as copA and copB expression. Hence, Yersinia has evolved a mechanism coupling active secretion of a plasmid-encoded component of the T3SS, YopD, to the regulation of plasmid replication. Our work provides evidence for the cross-talk between plasmid-encoded functions with the IncFII replicon.

Phenotypic Switch and Growth of Melanoma Spheroids in the Presence of Mast Cells: Potential Impact of Nutrient-starvation Effects.

BACKGROUND/AIM: Mast cells are abundant in melanoma tumors, and studies suggest that they can be either detrimental or protective for melanoma growth. However, the underlying mechanisms are not fully understood. MATERIALS AND METHODS: Here, we adopted an established hanging-drop spheroid system to investigate how mast cells influence melanoma growth and phenotype in a 3-D context. To address the underlying mechanism, we conducted transcriptomic and pathway analyses. RESULTS: In the presence of mast cells or mast cell-conditioned medium, growth of melanoma spheroids was profoundly reduced. Transcriptomic analysis revealed that mast cell-conditioned medium had extensive effects on the gene-expression patterns of melanoma. Pathway analyses revealed profound effects on the expression of genes related to amino acid and protein metabolism. The conditioned medium also induced up-regulation of cancer-related genes, including adhesion molecules implicated in metastatic spreading. In line with this, after transfer to a Matrigel extracellular matrix milieu, spheroids that had been developed in the presence of mast cell-conditioned medium displayed enhanced growth and adhesive properties. However, when assessing the possible impact of nutrient starvation, i.e., reduced nutrient content in mast cell-conditioned medium, we found that the observed effects on growth of melanoma spheroids could potentially be explained by such a scenario. CONCLUSION: Our findings suggest that the phenotypic alterations of melanoma spheroids grown in the presence of mast cells or mast cell-conditioned media are, at least partly, due to nutrient starvation rather than to the action of factors secreted by mast cells. Our findings may provide insight into the effects on gene-expression events that occur in melanoma tumors under nutrient stress.

Complete Genome Assembly of Multidrug-Resistant Yersinia enterocolitica Y72, Isolated in Sweden.

Here, we report the complete genome sequence of a Swedish clinical strain of Yersinia enterocolitica, Y72. With emerging Yersinia outbreaks circulating in Nordic countries, the Y72 genome will provide more insights on the genetic relatedness and antibiotic resistance dissemination in future studies.

Spatiotemporal Variations in Growth Rate and Virulence Plasmid Copy Number during Yersinia pseudotuberculosis Infection.

Pathogenic Yersinia spp. depend on the activity of a potent virulence plasmid-encoded ysc/yop type 3 secretion system (T3SS) to colonize hosts and cause disease. It was recently shown that Yersinia pseudotuberculosis upregulates the virulence plasmid copy number (PCN) during infection and that the resulting elevated gene dose of plasmid-encoded T3SS genes is essential for virulence. When and how this novel regulatory mechanism is deployed and regulates the replication of the virulence plasmid during infection is unknown. In the present study, we applied droplet digital PCR (ddPCR) to investigate the dynamics of Y. pseudotuberculosis virulence PCN variations and growth rates in infected mouse organs. We demonstrated that both PCN and growth varied in different tissues and over time throughout the course of infection, indicating that the bacteria adapted to discrete microenvironments during infection. The PCN was highest in Peyer's patches and cecum during the clonal invasive phase of the infection, while the highest growth rates were found in the draining mesenteric lymph nodes. In deeper, systemic organs, the PCN was lower and more modest growth rates were recorded. Our study indicates that increased gene dosage of the plasmid-encoded T3SS genes is most important early in the infection during invasion of the host. The described ddPCR approach will greatly simplify analyses of PCN, growth dynamics, and bacterial loads in infected tissues and will be readily applicable to other infection models.