Despite antiretroviral medications, the rate of pediatric HIV-1 infections through breast-milk transmission has been staggering in developing countries. Therefore, the development of a vaccine to protect vulnerable infant populations should be actively pursued. We previously demonstrated that oral immunization of newborn macaques with vesicular stomatitis virus expressing simian immunodeficiency virus genes

Critical to microbial versatility is the capacity to express the cohort of genes that increase fitness in different environments. Legionella pneumophila occupies extensive ecological space that includes diverse protists, pond water, engineered water systems, and mammalian lung macrophages. One mechanism that equips this opportunistic pathogen to adapt to fluctuating conditions is a switch between replicative and transmissive cell types that is controlled by the broadly conserved regulatory protein CsrA. A striking feature of the legionellae surveyed is that each of 14 strains encodes 4 to 7 csrA-like genes, candidate regulators of distinct fitness traits. Here we focus on the one csrA paralog (lpg1593) that, like the canonical csrA, is conserved in all 14 strains surveyed. Phenotypic analysis revealed that long-term survival in tap water is promoted by the lpg1593 locus, which we name csrR (for “CsrA-similar protein for resilience”). As predicted by its GGA motif, csrR mRNA was bound directly by the canonical CsrA protein, as judged by electromobility shift and RNA-footprinting assays. Furthermore, CsrA repressed translation of csrR mRNA in vivo, as determined by analysis of csrR-gfp reporters, csrR mRNA stability in the presence and absence of csrA expression, and mutation of the CsrA binding site identified on the csrR mRNA. Thus, CsrA not only governs the transition from replication to transmission but also represses translation of its paralog csrR when nutrients are available. We propose that, during prolonged starvation, relief of CsrA repression permits CsrR protein to coordinate L. pneumophila’s switch to a cell type that is resilient in water supplies.

To compare tissue-based pharmacokinetics and efficacy of oral tenofovir disoproxyl fumarate (TDF) versus subcutaneous tenofovir (TFV), macaques were treated for 2 weeks starting 1 week after simian immunodeficiency virus inoculation. Despite lower plasma TFV levels in the oral TDF arm, similar TFV diphosphate levels and antiviral activities were measured in lymphoid cells of most tissues. In intestinal tissues, however, oral TDF resulted in higher active drug levels, associated with lower virus levels and better immune preservation.

A 90-day repeated-dose oral toxicological evaluation was conducted according to GLP and OECD guidelines on lyophilized spores of the novel genetically modified strain B. subtilis ZB183. Lyophilized spores at doses of 109, 1010, and 1011 CFU/kg body weight/day were administered by oral gavage to Wistar rats for a period of 90 consecutive days. B. subtilis ZB183 had no effects on clinical signs, mortality, ophthalmological examinations, functional observational battery, body weights, body weight gains and food consumption in both sexes. There were no test item-related changes observed in haematology, coagulation, urinalysis, thyroid hormonal analysis, terminal fasting body weights, organ weights, gross pathology and histopathology. A minimal increase in the plasma albumin level was observed at 1010 and 1011 CFU/kg/day doses without an increase in total protein in males or females and was considered a nonadverse effect. The “No Observed Adverse Effect Level (NOAEL)” is defined at the highest dose of 1011 CFU/kg body weight/day for lyophilized B. subtilis ZB183 Spores under the test conditions employed.

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Bacterial evolution is accelerated by mobile genetic elements. To spread horizontally and to benefit the recipient bacteria, genes encoded on these elements must be properly regulated. Among the legionellae are multiple integrative conjugative elements (ICEs) that each encode a paralog of the broadly conserved regulator csrA. Using bioinformatic analyses, we deduced that specific csrA paralogs are coinherited with particular lineages of the type IV secretion system that mediates horizontal spread of its ICE, suggesting a conserved regulatory interaction. As a first step to investigate the contribution of csrA regulators to this class of mobile genetic elements, we analyzed here the activity of the csrA paralog encoded on Legionella pneumophila ICE-␤ox. Deletion of this gene, which we name csrT, had no observed effect under laboratory conditions. However, ectopic expression of csrT abrogated the protection to hydrogen peroxide and macrophage degradation that ICE-␤ox confers to L. pneumophila. When ectopically expressed, csrT also repressed L. pneumophila flagellin production and motility, a function similar to the core genome's canonical csrA. Moreover, csrT restored the repression of motility to csrA mutants of Bacillus subtilis, a finding consistent with the predicted function of CsrT as an mRNA binding protein. Since all known ICEs of legionellae encode coinherited csrA-type IV secretion system pairs, we postulate that CsrA superfamily proteins regulate ICE activity to increase their horizontal spread, thereby expanding L. pneumophila versatility. IMPORTANCEICEs are mobile DNA elements whose type IV secretion machineries mediate spread among bacterial populations. All surveyed ICEs within the Legionella genus also carry paralogs of the essential life cycle regulator csrA. It is striking that the csrA loci could be classified into distinct families based on either their sequence or the subtype of the adjacent type IV secretion system locus. To investigate whether ICE-encoded csrA paralogs are bona fide regulators, we analyzed ICE-␤ox as a model system. When expressed ectopically, its csrA paralog inhibited multiple ICE-␤ox phenotypes, as well as the motility of not only Legionella but also Bacillus subtilis. Accordingly, we predict that CsrA regulators equip legionellae ICEs to promote their spread via dedicated type IV secretion systems. Mobile DNA elements can spread advantageous traits among bacterial populations and contribute to the evolution of pathogens (1). Integrative conjugative elements (ICEs) are one type of mobile transposable element that encode their own type IV secretion system (T4SS) for transfer between strains and also the enzymes necessary to integrate the element into the host chromosome. ICEs have expanded the genetic repertoire of a number of bacterial pathogens by carrying a variety of cargo genes that confer advantageous traits for their hosts, enhancing fitness in specific environments. Some ICEs provide resistance to antibiotics, metal ions, and oxidative stress; others enhanc...

The ability to construct recombinant alleles efficiently in strains of interest, particularly unmarked deletions that reduce the potential for polar effects, is essential to studies of both pathogenesis and basic bacterial physiology. Here we describe a three-phase approach for generating unmarked deletions in Legionella pneumophila by constructing a mutant allele in E. coli using λ-Red recombination, so-called recombineering; transferring the allele onto the L. pneumophila chromosome by natural transformation; and then removing the selectable marker by utilizing the Flp site-specific recombinase. This strategy can decrease the amount of clone screening required while also increasing the percentage of the time the desired allele is obtained on the first attempt. The approach is particularly suited for constructing multiple unmarked deletions in a single strain in fewer steps than traditional methods.

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