The E. coli pathobiont LF82 encodes a unique variant of σ70 that results in specific gene expression changes and altered phenotypes.

LF82, an adherent invasive Escherichia coli pathobiont, is associated with ileal Crohn's disease, an inflammatory bowel disease of unknown etiology. Although LF82 contains no virulence genes, it carries several genetic differences, including single nucleotide polymorphisms (SNPs), that distinguish it from nonpathogenic E. coli. We have identified and investigated an extremely rare SNP that is within the highly conserved rpoD gene, encoding σ70, the primary sigma factor for RNA polymerase. We demonstrate that this single residue change (D445V) results in specific transcriptome and phenotypic changes that are consistent with multiple phenotypes observed in LF82, including increased antibiotic resistance and biofilm formation, modulation of motility, and increased capacity for methionine biosynthesis. Our work demonstrates that a single residue change within the bacterial primary sigma factor can lead to multiple alterations in gene expression and phenotypic changes, suggesting an underrecognized mechanism by which pathobionts and other strain variants with new phenotypes can emerge.

A phage-encoded nucleoid associated protein compacts both host and phage DNA and derepresses H-NS silencing.

Nucleoid Associated Proteins (NAPs) organize the bacterial chromosome within the nucleoid. The interaction of the NAP H-NS with DNA also represses specific host and xenogeneic genes. Previously, we showed that the bacteriophage T4 early protein MotB binds to DNA, co-purifies with H-NS/DNA, and improves phage fitness. Here we demonstrate using atomic force microscopy that MotB compacts the DNA with multiple MotB proteins at the center of the complex. These complexes differ from those observed with H-NS and other NAPs, but resemble those formed by the NAP-like proteins CbpA/Dps and yeast condensin. Fluorescent microscopy indicates that expression of motB in vivo, at levels like that during T4 infection, yields a significantly compacted nucleoid containing MotB and H-NS. motB overexpression dysregulates hundreds of host genes; ∼70% are within the hns regulon. In infected cells overexpressing motB, 33 T4 late genes are expressed early, and the T4 early gene repEB, involved in replication initiation, is up ∼5-fold. We postulate that MotB represents a phage-encoded NAP that aids infection in a previously unrecognized way. We speculate that MotB-induced compaction may generate more room for T4 replication/assembly and/or leads to beneficial global changes in host gene expression, including derepression of much of the hns regulon.

Effect of testosterone and estrogen supplementation on the resistance to systemic Candida albicans infection in mice.

Candida species are the 4th leading cause of nosocomial infections in the US affecting both men and women. Since males of many species can be more susceptible to infections than females, we investigated whether male mice were more susceptible to systemic Candida albicans (C. albicans) infection and if sex hormones were responsible for sex-dependent susceptibility to this infection. Non-gonadectomized or gonadectomized mice were supplemented with sustained release 5α-dihydrotestosterone (5αDHT) or 17-ß-estradiol (E2) using subcutaneous pellet implantation. Mice were challenged intravenously with 5 × 105 C. albicans/mouse seven days after pellet implantation and monitored for survival and weight change. We observed that male mice were more susceptible to systemic C. albicans infection than female mice while gonadectomized male mice were as resistant to the C. albicans infection as female mice. 5αDHT supplementation of gonadectomized female or male mice increased their susceptibility to the yeast infection while E2 supplementation of gonadectomized male mice did not increase their resistance to the infection. Overall, our results strongly suggest that testosterone plays an important role in decreasing resistance to systemic C. albicans infection.

The Bacteriophage T4 MotB Protein, a DNA-Binding Protein, Improves Phage Fitness.

The lytic bacteriophage T4 employs multiple phage-encoded early proteins to takeover the Escherichia coli host. However, the functions of many of these proteins are not known. In this study, we have characterized the T4 early gene motB, located in a dispensable region of the T4 genome. We show that heterologous production of MotB is highly toxic to E. coli, resulting in cell death or growth arrest depending on the strain and that the presence of motB increases T4 burst size 2-fold. Previous work suggested that motB affects middle gene expression, but our transcriptome analyses of T4 motBam vs. T4 wt infections reveal that only a few late genes are mildly impaired at 5 min post-infection, and expression of early and middle genes is unaffected. We find that MotB is a DNA-binding protein that binds both unmodified host and T4 modified [(glucosylated, hydroxymethylated-5 cytosine, (GHme-C)] DNA with no detectable sequence specificity. Interestingly, MotB copurifies with the host histone-like proteins, H-NS and StpA, either directly or through cobinding to DNA. We show that H-NS also binds modified T4 DNA and speculate that MotB may alter how H-NS interacts with T4 DNA, host DNA, or both, thereby improving the growth of the phage.

Effect of Delta-9-tetrahydrocannabinol on mouse resistance to systemic Candida albicans infection.

Delta-9-tetrahydrocannabinol (Δ9-THC), the psychoactive component of marijuana, is known to suppress the immune responses to bacterial, viral and protozoan infections, but its effects on fungal infections have not been studied. Therefore, we investigated the effects of chronic Δ9-THC treatment on mouse resistance to systemic Candida albicans (C. albicans) infection. To determine the outcome of chronic Δ9-THC treatment on primary, acute systemic candidiasis, c57BL/6 mice were given vehicle or Δ9-THC (16 mg/kg) in vehicle on days 1-4, 8-11 and 15-18. On day 19, mice were infected with 5×10(5) C. albicans. We also determined the effect of chronic Δ9-THC (4-64 mg/kg) treatment on mice infected with a non-lethal dose of 7.5×10(4) C. albicans on day 2, followed by a higher challenge with 5×10(5) C. albicans on day 19. Mouse resistance to the infection was assessed by survival and tissue fungal load. Serum cytokine levels were determine to evaluate the immune responses. In the acute infection, chronic Δ9-THC treatment had no effect on mouse survival or tissue fungal load when compared to vehicle treated mice. However, Δ9-THC significantly suppressed IL-12p70 and IL-12p40 as well as marginally suppressed IL-17 versus vehicle treated mice. In comparison, when mice were given a secondary yeast infection, Δ9-THC significantly decreased survival, increased tissue fungal burden and suppressed serum IFN-γ and IL-12p40 levels compared to vehicle treated mice. The data showed that chronic Δ9-THC treatment decreased the efficacy of the memory immune response to candida infection, which correlated with a decrease in IFN-γ that was only observed after the secondary candida challenge.