Identification of a novel zinc metalloprotease through a global analysis of clostridium difficile extracellular proteins

Clostridium difficile is a major cause of infectious diarrhea worldwide. Although the cell surface proteins are recognized to be important in clostridial pathogenesis, biological functions of only a few are known. Also, apart from the toxins, proteins exported by C. difficile into the extracellular milieu have been poorly studied. In order to identify novel extracellular factors of C. difficile, we analyzed bacterial culture supernatants prepared from clinical isolates, 630 and R20291, using liquid chromatography-tandem mass spectrometry. The majority of the proteins identified were non-canonical extracellular proteins. These could be largely classified into proteins associated to the cell wall (including CWPs and extracellular hydrolases), transporters and flagellar proteins. Seven unknown hypothetical proteins were also identified. One of these proteins, CD630_28300, shared sequence similarity with the anthrax lethal factor, a known zinc metallopeptidase. We demonstrated that CD630_28300 (named Zmp1) binds zinc and is able to cleave fibronectin and fibrinogen in vitro in a zinc-dependent manner. Using site-directed mutagenesis, we identified residues important in zinc binding and enzymatic activity. Furthermore, we demonstrated that Zmp1 destabilizes the fibronectin network produced by human fibroblasts. Thus, by analyzing the exoproteome of C. difficile, we identified a novel extracellular metalloprotease that may be important in key steps of clostridial pathogenesis

Clostridium difficile infection.

Infection of the colon with the Gram-positive bacterium Clostridium difficile is potentially life threatening, especially in elderly people and in patients who have dysbiosis of the gut microbiota following antimicrobial drug exposure. C. difficile is the leading cause of health-care-associated infective diarrhoea. The life cycle of C. difficile is influenced by antimicrobial agents, the host immune system, and the host microbiota and its associated metabolites. The primary mediators of inflammation in C. difficile infection (CDI) are large clostridial toxins, toxin A (TcdA) and toxin B (TcdB), and, in some bacterial strains, the binary toxin CDT. The toxins trigger a complex cascade of host cellular responses to cause diarrhoea, inflammation and tissue necrosis - the major symptoms of CDI. The factors responsible for the epidemic of some C. difficile strains are poorly understood. Recurrent infections are common and can be debilitating. Toxin detection for diagnosis is important for accurate epidemiological study, and for optimal management and prevention strategies. Infections are commonly treated with specific antimicrobial agents, but faecal microbiota transplants have shown promise for recurrent infections. Future biotherapies for C. difficile infections are likely to involve defined combinations of key gut microbiota

Stag beetle battle behavior and its associated anatomical adaptations

Male stag beetles battle for females with their impressive, oversized mandibles. We describe their fighting behavior, which is essential to understand the evolution and morphology of their weaponry. Our behavioral analysis reveals several anatomical structures that are important for fighting, and our morphological investigations show how these may be adapted for their functions. Stag beetle fights are much more variable than other armed beetles' battles. They spend considerable time and effort in dislodging their opponent, that clings to the substrate with its tarsal claws. These tarsal claws are also indispensable to maintain balance in the most spectacular battles, when they lift a rival high in the air. The male claws are highly curved and have an increased height for this purpose. The prothoracic muscles are hypertrophied to support the lifting movement. The largest beetle wins in 85 % of the fights and the smaller the difference in mandible length is between the rivals, the longer the battles can last. The long mandibles enable males to reach the opponent's legs in order to dislodge it. For this purpose, they bite with all parts of their mandibles, even though the distal part is more vulnerable for failure and transfers less bite force. Blindfolded experiments prove that visual information is not a requisite for a successful battle

Morphological Variation of the Forelimb and Claw in Neotropical Sigmodontine Rodents (Rodentia: Cricetidae)

The limbs of mammals exhibit a variety of morphologies that reflect the diversity of their habitats and their functional needs, including subtle structural differences in their distal limb integumentary appendages (hooks, claws, adhesive pads). Little is known about structure and function of claws of sigmodontine rodents. Here, we analyze claw shape and forelimb skeleton morphology of 25 species of sigmodontine rodents with different locomotory types (ambulatory, fossorial, natatorial, quadrupedal saltatorial, and scansorial), taking into account their phylogenetic affinities. Qualitative differences in claw shape were examined using digital photographs, and quantitative measurements were made for length, height, and curvature of the claws of all digits, and dimensions of other forelimb skeletal elements. Our results show that both phylogeny and ecological categories explain substantial components of the morphological variation in sigmodontine rodents. Qualitative analysis reveals that non-specialized forms (ambulatory, quadrupedal saltatorial, and scansorial) tend to have high and strongly curved claws, whereas highly specialized forms (fossorial and natatorial) tend to have elongate and smoothly curved claws. However, the quantitative analysis differentiated the fossorial and scansorial by variables related to claw, and natatorial by variables related to bones of the forelimb. No variables that could differentiate ambulatory or quadrupedal saltatorial forms were found, demonstrating that these forms show a generalized morphological pattern. This study indicates that both historical and ecological factors contribute to the evolution of claw length in these groups.Fil: Tulli, María José. Fundación Miguel Lillo. Dirección de Zoología. Instituto de Herpetología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Carrizo, Luz Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas; Argentina. Universidad Nacional de Misiones; ArgentinaFil: Samuels, J. X.. John Day Fossil Beds National Monument; Estados Unido