Highly efficient production of the staphylococcal nuclease reporter in Lactobacillus bulgaricus governed by the promoter of the hlbA gene

Lactobacillus delbrueckii ssp. bulgaricus ( L. bulgaricus ) genome sequence analysis revealed the presence of two genes that encode histone-like HU proteins ( hlbA and hlbB ) showing extensive similarity to other bacterial homologues. These genes were found to be extremely conserved among several L. bulgaricus strains. The hlbA gene was shown to be constitutively transcribed from a unique promoter ( phlbA ) during normal growth, whereas hlbB did not seem to be expressed under usual laboratory conditions. Using a reporter cassette in which the staphylococcal nuclease was fused at its N-terminus to the lactococcal signal peptide Usp45 (SP Usp45), we have demonstrated that phlbA promotes high expression of the reporter in L. bulgaricus , which correlated with an abundant secretion of the mature nuclease in the supernatant fraction. Quantification of the exported enzyme reveals a secretion level approximately threefold higher when the expression of the reporter was under the control of phlbA compared with the lactococcal usp45 promoter. Together, these results indicate that phlbA is suitable for gene expression in L. bulgaricus , that SP Usp45 is functionally recognized and processed by the L. bulgaricus secretion machinery and that the nuclease reporter gene can be used for the identification of exported products in this bacterium.     

Team swimming in ant spermatozoa

In species where females mate promiscuously, competition between ejaculates from different males to fertilize the ova is an important selective force shaping many aspects of male reproductive traits, such as sperm number, sperm length and sperm–sperm interactions. In eusocial Hymenoptera (bees, wasps and ants), males die shortly after mating and their reproductive success is ultimately limited by the amount of sperm stored in the queen''s spermatheca. Multiple mating by queens is expected to impose intense selective pressure on males to optimize the transfer of sperm to the storage organ. Here, we report a remarkable case of cooperation between spermatozoa in the desert ant Cataglyphis savignyi. Males ejaculate bundles of 50–100 spermatozoa. Sperm bundles swim on average 51% faster than solitary sperm cells. Team swimming is expected to increase the amount of sperm stored in the queen spermatheca and, ultimately, enhance male posthumous fitness.     

Endocytosis of Viruses and Bacteria

Of the many pathogens that infect humans and animals, a large number use cells of the host organism as protected sites for replication. To reach the relevant intracellular compartments, they take advantage of the endocytosis machinery and exploit the network of endocytic organelles for penetration into the cytosol or as sites of replication. In this review, we discuss the endocytic entry processes used by viruses and bacteria and compare the strategies used by these dissimilar classes of pathogens.Many of the most widespread and devastating diseases in humans and livestock are caused by viruses and bacteria that enter cells for replication. Being obligate intracellular parasites, viruses have no choice. They must transport their genome to the cytosol or nucleus of infected cells to multiply and generate progeny. Bacteria and eukaryotic parasites do have other options; most of them can replicate on their own. However, some have evolved to take advantage of the protected environment in the cytosol or in cytoplasmic vacuoles of animal cells as a niche favorable for growth and multiplication. In both cases (viruses and intracellular bacteria), the outcome is often destructive for the host cell and host organism. The mortality and morbidity caused by infectious diseases worldwide provide a strong rationale for research into pathogen–host cell interactions and for pursuing the detailed mechanisms of transmission and dissemination. The study of viruses and bacteria can, moreover, provide invaluable insights into fundamental aspects of cell biology.Here, we focus on the mechanisms by which viral and bacterial pathogens exploit the endocytosis machinery for host cell entry and replication. Among recent reviews on this topic, dedicated uniquely to either mammalian viruses or bacterial pathogens, we recommend the following: Cossart and Sansonetti (2004); Pizarro-Cerda and Cossart (2006); Kumar and Valdivia (2009); Cossart and Roy (2010); Mercer et al. (2010b); Grove and Marsh (2011); Kubo et al. (2012); Vazquez-Calvo et al. (2012a); Sun et al. (2013).The term “endocytosis” is used herein in its widest sense, that is, to cover all processes whereby fluid, solutes, ligands, and components of the plasma membrane as well as particles (including pathogenic agents) are internalized by cells through the invagination of the plasma membrane and the scission of membrane vesicles or vacuoles. This differs from current practice in the bacterial pathogenesis field, where the term “endocytosis” is generally reserved for the internalization of molecules or small objects, whereas the uptake of bacteria into nonprofessional phagocytes is called “internalization” or “bacterial-induced phagocytosis.” In addition, the term “phagocytosis” is reserved for internalization of bacteria by professional phagocytes (macrophages, polymorphonuclear leucocytes, dendritic cells, and amoebae), a process that generally but not always leads to the destruction of the ingested bacteria (Swanson et al. 1999; May and Machesky 2001; Henry et al. 2004; Zhang et al. 2010). With a few exceptions, we will not discuss phagocytosis of bacteria or the endocytosis of protozoan parasites such as Toxoplasma and Plasmodium (Robibaro et al. 2001).     

The Staphylococcus aureus RNome and its commitment to virulence

Staphylococcus aureus is a major human pathogen causing a wide spectrum of nosocomial and community-associated infections with high morbidity and mortality. S. aureus generates a large number of virulence factors whose timing and expression levels are precisely tuned by regulatory proteins and RNAs. The aptitude of bacteria to use RNAs to rapidly modify gene expression, including virulence factors in response to stress or environmental changes, and to survive in a host is an evolving concept. Here, we focus on the recently inventoried S. aureus regulatory RNAs, with emphasis on those with identified functions, two of which are directly involved in pathogenicity.     

Reliability of recording uterine cancer in death certification in France and age-specific proportions of deaths from cervix and corpus uteri

French uterine cancer recordings in death certificates include 60% of "uterine cancer, Not Otherwise Specified (NOS)"; this hampers the estimation of mortalities from cervix and corpus uteri cancers. The aims of this work were to study the reliability of uterine cancer recordings in death certificates using a case matching with cancer registries and estimate age-specific proportions of deaths from cervix and corpus uteri cancers among all uterine cancer deaths by a statistical approach that uses incidence and survival data. Deaths from uterine cancer between 1989 and 2001 were extracted from the French National database of causes of death and case-to-case matched to women diagnosed with uterine cancer between 1989 and 1997 in 8 cancer registries. Registry data were considered as "gold-standard". Among the 1825 matched deaths, cancer registries recorded 830 cervix and 995 corpus uteri cancers. In death certificates, 5% and 40% of "true" cervix cancers were respectively coded "corpus" and "uterus, NOS" and 5% and 59% of "true" corpus cancers respectively coded "cervix" and "uterus, NOS". Miscoding cervix cancers was more frequent at advanced ages at death and in deaths at home or in small urban areas. Miscoding corpus cancers was more frequent in deaths at home or in small urban areas. From the statistical method, the estimated proportion of deaths from cervix cancer among all uterine cancer deaths was higher than 95% in women aged 30-40 years old but declined to 35% in women older than 70 years. The study clarifies the reason for poor encoding of uterus cancer mortality and refines the estimation of mortalities from cervix and corpus uteri cancers allowing future studies on the efficacy of cervical cancer screening.     

Characterization of a novel type of HIV-1 particle assembly inhibitor using a quantitative luciferase-Vpr packaging-based assay

The HIV-1 auxiliary protein Vpr and Vpr-fusion proteins can be copackaged with Gag precursor (Pr55Gag) into virions or membrane-enveloped virus-like particles (VLP). Taking advantage of this property, we developed a simple and sensitive method to evaluate potential inhibitors of HIV-1 assembly in a living cell system. Two proteins were coexpressed in recombinant baculovirus-infected Sf9 cells, Pr55Gag, which formed the VLP backbone, and luciferase fused to the N-terminus of Vpr (LucVpr). VLP-encapsidated LucVpr retained the enzymatic activity of free luciferase. The levels of luciferase activity present in the pelletable fraction recovered from the culture medium correlated with the amounts of extracellular VLP released by Sf9 cells assayed by conventional immunological methods. Our luciferase-based assay was then applied to the characterization of betulinic acid (BA) derivatives that differed from the leader compound PA-457 (or DSB) by their substituant on carbon-28. The beta-alanine-conjugated and lysine-conjugated DSB could not be evaluated for their antiviral potentials due to their high cytotoxicity, whereas two other compounds with a lesser cytotoxicity, glycine-conjugated and ε-NH-Boc-lysine-conjugated DSB, exerted a dose-dependent negative effect on VLP assembly and budding. A fifth compound with a low cytotoxicity, EP-39 (ethylene diamine-conjugated DSB), showed a novel type of antiviral effect. EP-39 provoked an aberrant assembly of VLP, resulting in nonenveloped, morula-like particles of 100-nm in diameter. Each morula was composed of nanoparticle subunits of 20-nm in diameter, which possibly mimicked transient intermediates of the HIV-1 Gag assembly process. Chemical cross-linking in situ suggested that EP-39 favored the formation or/and persistence of Pr55Gag trimers over other oligomeric species. EP-39 showed a novel type of negative effect on HIV-1 assembly, targeting the Pr55Gag oligomerisation. The biological effect of EP-39 underlined the critical role of the nature of the side chain at position 28 of BA derivatives in their anti-HIV-1 activity.