Cell-Wall Hydrolases as Antimicrobials against Staphylococcus Species: Focus on Sle1.

Some staphylococcal species are opportunistic pathogens of humans and/or animals with Staphylococcus epidermidis as one of the most important. It causes a broad spectrum of diseases in humans and animals. This species is able to form biofilms and has developed antibiotic resistance, which has motivated research on new antibacterial agents. Cell-wall hydrolases (CWHs) can constitute a potential alternative. Following a hijacking strategy, we inventoried the CWHs of S. epidermidis. The lytic potential of representative CWHs that could be turned against staphylococci was explored by turbidity assays which revealed that cell wall glycosidases were not efficient, while cell wall amidases and cell wall peptidases were able to lyse S. epidermidis. Sle1, which is encoded by chromosomal gene and composed of three anchoring LysM domains and a C-terminal CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain, was one of the most active CWHs. The phylogeny of Sle1 revealed seven clusters mostly identified among staphylococci. Sle1 was able to lyse several staphylococcal species, including Staphylococcus aureus, both in planktonic and sessile forms, but not Micrococcus.

Cell Wall Hydrolases in Bacteria: Insight on the Diversity of Cell Wall Amidases, Glycosidases and Peptidases Toward Peptidoglycan.

The cell wall (CW) of bacteria is an intricate arrangement of macromolecules, at least constituted of peptidoglycan (PG) but also of (lipo)teichoic acids, various polysaccharides, polyglutamate and/or proteins. During bacterial growth and division, there is a constant balance between CW degradation and biosynthesis. The CW is remodeled by bacterial hydrolases, whose activities are carefully regulated to maintain cell integrity or lead to bacterial death. Each cell wall hydrolase (CWH) has a specific role regarding the PG: (i) cell wall amidase (CWA) cleaves the amide bond between N-acetylmuramic acid and L-alanine residue at the N-terminal of the stem peptide, (ii) cell wall glycosidase (CWG) catalyses the hydrolysis of the glycosidic linkages, whereas (iii) cell wall peptidase (CWP) cleaves amide bonds between amino acids within the PG chain. After an exhaustive overview of all known conserved catalytic domains responsible for CWA, CWG, and CWP activities, this review stresses that the CWHs frequently display a modular architecture combining multiple and/or different catalytic domains, including some lytic transglycosylases as well as CW binding domains. From there, direct physiological and collateral roles of CWHs in bacterial cells are further discussed.

Insight into the Genome of Staphylococcus xylosus, a Ubiquitous Species Well Adapted to Meat Products.

Staphylococcus xylosus belongs to the vast group of coagulase-negative staphylococci. It is frequently isolated from meat products, either fermented or salted and dried, and is commonly used as starter cultures in sausage manufacturing. Analysis of the S. xylosus genome together with expression in situ in a meat model revealed that this bacterium is well adapted to meat substrates, being able to use diverse substrates as sources of carbon and energy and different sources of nitrogen. It is well-equipped with genes involved in osmotic, oxidative/nitrosative, and acidic stress responses. It is responsible for the development of the typical colour of cured meat products via its nitrate reductase activity. It contributes to sensorial properties, mainly by the the catabolism of pyruvate and amino acids resulting in odorous compounds and by the limiting of the oxidation of fatty acids, thereby avoiding rancidity.

Ferritin, an iron source in meat for Staphylococcus xylosus?

Staphylococcus xylosus is frequently isolated from food of animal origin. Moreover, this species is one of the major starter cultures used for meat fermentation. Iron is a key element for growth and survival of bacteria. Meat is particularly rich in haemic (myoglobin and haemoglobin) and non-haemic (ferritin and transferrin) iron sources. Ferritin is a storage protein able to capture large quantities of iron. It is highly resistant to microbial attack and few microorganisms can use it as an iron source. Surprisingly, we found that the S. xylosus C2a strain grows in the presence of ferritin as a sole iron source. A three-cistron operon was highly overexpressed under ferritin iron growth conditions. We generated a deletion-insertion in the first gene of the operon and evaluated the phenotype of the mutant. The mutant showed decreased growth because it was less able to acquire iron from ferritin. Transcriptional analysis of the mutant revealed downregulation of several genes involved in the response to oxidative stress. This study characterized for the first time the capacity of a Staphylococcus to use iron from ferritin and revealed that a potential reductive pathway was involved in this acquisition. We hypothesize that this ability could give an advantage to S. xylosus in meat products.

Adaptation of Staphylococcus xylosus to Nutrients and Osmotic Stress in a Salted Meat Model.

Staphylococcus xylosus is commonly used as starter culture for meat fermentation. Its technological properties are mainly characterized in vitro, but the molecular mechanisms for its adaptation to meat remain unknown. A global transcriptomic approach was used to determine these mechanisms. S. xylosus modulated the expression of about 40-50% of the total genes during its growth and survival in the meat model. The expression of many genes involved in DNA machinery and cell division, but also in cell lysis, was up-regulated. Considering that the S. xylosus population remained almost stable between 24 and 72 h of incubation, our results suggest a balance between cell division and cell lysis in the meat model. The expression of many genes encoding enzymes involved in glucose and lactate catabolism was up-regulated and revealed that glucose and lactate were used simultaneously. S. xylosus seemed to adapt to anaerobic conditions as revealed by the overexpression of two regulatory systems and several genes encoding cofactors required for respiration. In parallel, genes encoding transport of peptides and peptidases that could furnish amino acids were up-regulated and thus concomitantly a lot of genes involved in amino acid synthesis were down-regulated. Several genes involved in glutamate homeostasis were up-regulated. Finally, S. xylosus responded to the osmotic stress generated by salt added to the meat model by overexpressing genes involved in transport and synthesis of osmoprotectants, and Na(+) and H(+) extrusion.

Transcriptomic analysis of Staphylococcus xylosus in the presence of nitrate and nitrite in meat reveals its response to nitrosative stress.

Staphylococcus xylosus is one of the major starter cultures used for meat fermentation because of its crucial role in the reduction of nitrate to nitrite which contributes to color and flavor development. Despite longstanding use of these additives, their impact on the physiology of S. xylosus has not yet been explored. We present the first in situ global gene expression profile of S. xylosus in meat supplemented with nitrate and nitrite at the levels used in the meat industry. More than 600 genes of S. xylosus were differentially expressed at 24 or 72 h of incubation. They represent more than 20% of the total genes and let us to suppose that addition of nitrate and nitrite to meat leads to a global change in gene expression. This profile revealed that S. xylosus is subject to nitrosative stress caused by reactive nitrogen species (RNS) generated from nitrate and nitrite. To overcome this stress, S. xylosus has developed several oxidative stress resistance mechanisms, such as modulation of the expression of several genes involved in iron homeostasis and in antioxidant defense. Most of which belong to the Fur and PerR regulons, respectively. S. xylosus has also counteracted this stress by developing DNA and protein repair. Furthermore, it has adapted its metabolic response-carbon and nitrogen metabolism, energy production and cell wall biogenesis-to the alterations produced by nitrosative stress.