Identification and characterization of a novel lytic peptidoglycan transglycosylase (MltC) in Shigella dysenteriae.

Shigellosis remains a worldwide health problem due to the lack of vaccines and the emergence of antibiotic-resistant strains. Shigella (S.) dysenteriae has rigid peptidoglycan (PG), and its tight regulation of biosynthesis and remodeling is essential for bacterial integrity. Lytic transglycosylases are highly conserved PG autolysins in bacteria that play essential roles in bacterial growth. However, their precise functions are obscure. We aimed to identify, clone, and express MltC, a unique autolysin in Escherichia (E.) coli C41 strain. The purification of recombinant MltC protein was performed using affinity chromatography and size-exclusion chromatography methods. The PG enzymatic activity of MltC was investigated using Zymogram and Fluorescein isothiocyanate (FITC)-labeled PG assays. Also, we aimed to detect its localization in bacterial fractions (cytoplasm and membrane) by western blot using specific polyclonal anti-MltC antibodies and its probable partners using immunoprecipitation and mass spectrometry applications. Purified MltC showed autolysin activity. Native MltC showed various locations in S. dysenteriae cells during different growth phases. In the Lag and early stationary phases, MltC was not found in cytoplasm and membrane fractions. However, it was detected in cytoplasm and membrane fractions during the exponential phase. In the late stationary phase, MltC was expressed in the membrane fraction only. Different candidate protein partners of MltC were identified that could be essential for bacterial growth and pathogenicity. This is the first study to suggest that MltC is indeed autolysin and could be a new drug target for the treatment of shigellosis by understanding its biological functions.

Biochemical characterization of LysVpKK5 endolysin from a marine vibriophage.

Endolysins have been proposed as a potential antibacterial alternative for aquaculture, especially against Vibrio; the bacterial-agents that most frequently cause disease. Although multiple marine vibriophages have been characterized to date, research on vibriophage endolysins is recent. In this study, biochemical characterization of LysVpKK5 endolysin encoded by Vibrio parahaemolyticus-infecting VpKK5 phage was performed. In silico analysis revealed that LysVpKK5 possesses a conserved amidase_2 domain with a zinc-binding motif of high structural similarity to T7 lysozyme (RMSD = 0.107 Å). Contrary to expectations, the activity was inhibited with Zn2+ and was improved with other divalent cations, especially Ca2+. It showed optimal muralytic activity at pH 10, and curiously, no lytic activity at pH ≤ 7 was recorded. As for the thermal stability test, the optimal activity was recorded at 30 °C; the higher residual activity was recorded at 4 °C, and was lost at ≥ 50 °C. On the other hand, increasing NaCl concentrations reduced the activity gradually; the optimal activity was recorded at 50 mM NaCl. On the other hand, the enzymatic activity at 0.5 M NaCl was approx 30% and of approx 50% in seawater. LysVpKK5 endolysin exhibited a higher activity on V. parahaemolyticus ATCC-17802 strain, in comparison with AHPND + strains.

Murein hydrolase activity in the surface layer of Lactobacillus acidophilus ATCC 4356.

We describe a new enzymatic functionality for the surface layer (S-layer) of Lactobacillus acidophilus ATCC 4356, namely, an endopeptidase activity against the cell wall of Salmonella enterica serovar Newport, assayed via zymograms and identified by Western blotting. Based on amino acid sequence comparisons, the hydrolase activity was predicted to be located at the C terminus. Subsequent cloning and expression of the C-terminal domain in Bacillus subtilis resulted in the functional verification of the enzymatic activity.

Pneumococcal whole-cell vaccine: optimization of cell growth of unencapsulated Streptococcus pneumoniae in bioreactor using animal-free medium.

The high cost of the available pneumococcal conjugated vaccines has been an obstacle in implementing vaccination programs for children in developing countries. As an alternative, Malley et al. proposed a vaccine consisting of inactivated whole-cells of unencapsulated S. pneumoniae, which provides serotype-independent protection and involves lower production costs. Although the pneumococcus has been extensively studied, little research has focused on its large-scale culture, thus implying a lack of knowledge of process parameters, which in turn are essential for its successful industrial production. The strain Rx1Al- eryR was originally cultured in Todd-Hewitt medium (THY), which is normally used for pneumococcus isolation, but is unsuitable for human vaccine preparations. The purposes of this study were to compare the strains Rx1Al- eryR and kanR, develop a new medium, and generate new data parameters for scaling-up the process. In static flasks, cell densities were higher for eryR than kanR. In contrast, the optical density (OD) of the former decreased immediately after reaching the stationary phase, and the OD of the latter remained stable. The strain Rx1Al- kanR was cultivated in bioreactors with medium based on either acid-hydrolyzed casein (AHC) or enzymatically hydrolyzed soybean meal (EHS). Biomass production in EHS was 2.5 times higher than in AHC, and about ten times higher than in THY. The process developed for growing the strain Rx1Al- kanR in pH-controlled bioreactors was shown to be satisfactory to this fastidious bacterium. The new culture conditions using this animal-free medium may allow the production of the pneumococcal whole-cell vaccine.

Peptidoglycan hydrolases in an autolytic mutant of Salmonella typhimurium.

Two peptidoglycan hydrolases were isolated from the autolytic mutant Salmonella typhimurium DA361 (envD). One of them, resistant to penicillin, was found free in the supernatant of partially purified envelopes sedimented by ultracentrifugation, and the other bound to the envelopes proved to be sensitive to the antibiotic. Both were able to hydrolyse in vitro high molecular weight non-specific peptidoglycan isolated from E. coli W7 labelled with [14C]diaminopimelic acid. Similar enzymatic activities were separated also from S. typhimurium DA362 (envD+) a non-lytic isogenic pair of the above and from the wild type strain LT-2. All of the hydrolytic activities reported here were strongly inhibited when DNA was added to the assay systems. The peptidoglycan hydrolases isolated from the autolytic mutant suffered a competitive inhibition while those from the non-lytic strains were apparently inhibited in uncompetitive modal relationship. It is postulated that the inhibitory effect may bear affinity with the preservation of DNA sites of attachment to cell membranes sustaining peptidoglycan structure and functions.