Requirement of the CXXC motif of novel Francisella infectivity potentiator protein B FipB, and FipA in virulence of F. tularensis subsp. tularensis

The lipoprotein encoded by the Francisella tularensis subsp. tularensis locus FTT1103 is essential for virulence; an FTT1103 deletion mutant is defective in uptake and intracellular survival, and mice survive high dose challenges of greater than 10(8) bacteria. This protein has two conserved domains; one is found in a class of virulence proteins called macrophage infectivity potentiator (Mip) proteins, and the other in oxidoreductase Disulfide Bond formation protein A (DsbA)-related proteins. We have designated the protein encoded by FTT1103 as FipB for Francisellainfectivity potentiator protein B. The locus FTT1102 (fipA), which is upstream of fipB, also has similarity to same conserved Mip domain. Deletion and site-specific mutants of fipA and fipB were constructed in the Schu S4 strain, and characterized with respect to intracellular replication and in vivo virulence. A nonpolar fipA mutant demonstrated reduced survival in host cells, but was only slightly attenuated in vivo. Although FipB protein was present in a fipA mutant, the abundance of the three isoforms of FipB was altered, suggesting that FipA has a role in post-translational modification of FipB. Similar to many DsbA homologues, FipB contains a cysteine-any amino acid-any amino acid-cysteine (CXXC) motif. This motif was found to be important for FipB's role in virulence; a deletion mutant complemented with a gene encoding a FipB protein in which the first cysteine was changed to an alanine residue (AXXC) failed to restore intracellular survival or in vivo virulence. Complementation with a gene that encoded a CXXA containing FipB protein was significantly defective in intracellular growth; however, only slightly attenuated in vivo.     

Proteus mirabilis interkingdom swarming signals attract blow flies

Flies transport specific bacteria with their larvae that provide a wider range of nutrients for those bacteria. Our hypothesis was that this symbiotic interaction may depend on interkingdom signaling. We obtained Proteus mirabilis from the salivary glands of the blow fly Lucilia sericata; this strain swarmed significantly and produced a strong odor that attracts blow flies. To identify the putative interkingdom signals for the bacterium and flies, we reasoned that as swarming is used by this bacterium to cover the food resource and requires bacterial signaling, the same bacterial signals used for swarming may be used to communicate with blow flies. Using transposon mutagenesis, we identified six novel genes for swarming (ureR, fis, hybG, zapB, fadE and PROSTU_03490), then, confirming our hypothesis, we discovered that fly attractants, lactic acid, phenol, NaOH, KOH and ammonia, restore swarming for cells with the swarming mutations. Hence, compounds produced by the bacterium that attract flies also are utilized for swarming. In addition, bacteria with the swarming mutation rfaL attracted fewer blow flies and reduced the number of eggs laid by the flies. Therefore, we have identified several interkingdom signals between P. mirabilis and blow flies.     

A comparison of assisted and unassisted proprioceptive neuromuscular facilitation techniques and static stretching

A comparison of assisted and unassisted proprioceptive neuromuscular facilitation techniques and static stretching. J Strength Cond Res 26(5): 1238-1244, 2012-Proprioceptive neuromuscular facilitation (PNF) stretching often requires a partner. Straps are available allowing an individual to perform PNF stretching alone. It is not known if a strap provides similar improvements in the range of motion (ROM) as partner-assisted PNF or static stretching. The purpose of this study was to compare assisted and unassisted (with a strap) PNF stretching and static stretching. Hip joint ROM, reaction time (RT), and movement time (MT) were measured prestretching and poststretching. Thirteen recreationally active adults participated in this study. The participants were subjected to 5 different stretch interventions in a random order on separate days. Stretch conditions included unassisted PNF stretching using (a) isometric, (b) concentric, and (c) eccentric contractions with a stretch strap, (d) partner-assisted isometric PNF, and (e) static stretching. The RT, MT, dynamic, active, passive hip flexion angle, and angular velocity with dynamic hip flexion were measured before and after the intervention. The ROM improved (p < 0.05) 2.6, 2.7, and 5.4%, respectively, with dynamic, active static, and passive static ROM, but there was no significant difference between the stretching protocols. There was a main effect for time (p < 0.05) with all stretching conditions negatively impacting dynamic angular velocity (9.2%). Although there was no significant effect on RT, MT showed a negative main effect for time (p < 0.05) slowing 3.4%. In conclusion, it was found that all 3 forms of active stretching provided similar improvements in the ROM and poststretching performance decrements in MT and angular velocity. Thus, individuals can implement PNF stretching techniques with a partner or alone with a strap to improve ROM, but athletes should not use these techniques before important competitions or training because of the impairment of limb velocity and MT.     

Identification of super-infected Aedes triseriatus mosquitoes collected as eggs from the field and partial characterization of the infecting La Crosse viruses

Background

La Crosse virus (LACV) is a pathogenic arbovirus that is transovarially transmitted by Aedes triseriatus mosquitoes and overwinters in diapausing eggs. However, previous models predicted transovarial transmission (TOT) to be insufficient to maintain LACV in nature.

Results

To investigate this issue, we reared mosquitoes from field-collected eggs and assayed adults individually for LACV antigen, viral RNA by RT-PCR, and infectious virus. The mosquitoes had three distinct infection phenotypes: 1) super infected (SI+) mosquitoes contained infectious virus, large accumulations of viral antigen and RNA and comprised 17 of 17,825 (0.09%) of assayed mosquitoes, 2) infected mosquitoes (I+) contained no detectable infectious virus, lesser amounts of viral antigen and RNA, and comprised 3.7% of mosquitoes, and 3) non-infected mosquitoes (I-) contained no detectable viral antigen, RNA, or infectious virus and comprised 96.21% of mosquitoes. SI+ mosquitoes were recovered in consecutive years at one field site, suggesting that lineages of TOT stably-infected and geographically isolated Ae. triseriatus exist in nature. Analyses of LACV genomes showed that SI+ isolates are not monophyletic nor phylogenetically distinct and that synonymous substitution rates exceed replacement rates in all genes and isolates. Analysis of singleton versus shared mutations (Fu and Li's F*) revealed that the SI+ LACV M segment, with a large and significant excess of intermediate-frequency alleles, evolves through disruptive selection that maintains SI+ alleles at higher frequencies than the average mutation rate. A QTN in the LACV NSm gene was detected in SI+ mosquitoes, but not in I+ mosquitoes. Four amino acid changes were detected in the LACV NSm gene from SI+ but not I+ mosquitoes from one site, and may condition vector super infection. In contrast to NSm, the NSs sequences of LACV from SI+ and I+ mosquitoes were identical.

Conclusions

SI+ mosquitoes may represent stabilized infections of Ae. triseriatus mosquitoes, which could maintain LACV in nature. A gene-for-gene interaction involving the viral NSm gene and a vector innate immune response gene may condition stabilized infection.     

Molecular structure of human fibroblast and leukocyte interferons: probe by lectin and hydrophobic chromatography.

Structural differences between human leukocyte virus-induced interferon and human fibroblast polyinosinic-polycytidylic acid (rIn-rCn)-induced interferon have been noted in previous studies. This study reports the behavior of human leukocyte and fibroblast interferon, induced by virus and by rIn-rCn, in several lectin and hydrophobic chromatographic systems. Differences in both glycosylation and in hydrophobicity of human leukocyte and fibroblast interferons are documented. Human fibroblast interferon is a glycoprotein, whereas our evidence suggests that human leukocyte interferon probably is not. Also, fibroblast interferon is more hydrophobic than leukocyte interferon, as probed on several hydrophobic adsorbents. The possible relationships of these differences to each other and to antigenic variations are discussed. Generally, the differences appear to be attributable to the cell type in which the interferon was induced. However, our results suggest that at least subtle differences in the processing of the induction signal (virus or rIn-rCn) within the same cell type may occur, slightly altering some structural features.     

Screening microbiota for effects on host tissues

The assembly and function of microbial communities depends on many factors including the local environment and the metabolic properties of the colonizing organisms. Chemical communications or other secreted factors also play a role and are used by different microbial strains both cooperatively and competitively. The spectrum of microbial secretions have various effects on the microbe's respective hosts, both positive and negative. Thus, characterizing the roles of microbial community members and their secretions can yield key mechanistic insights into microbiome function and can lead to new intervention strategies. Focusing on the simple, yet important functional impact of toxicity, we quantify supernatant dosage responses with image data and examine the morphological effects of microbial secretions on skin-associated host cells. Since the diversity of microbial communities, coupled with the multiplicity of host tissues requires scalable methods, we develop and demonstrate a microfluidic device that enables high-content screening of microbial secretion effects on adherent cell types.