Microbiological research最新文献

Brucella relies on the type IV secretion system (T4SS) to establish replication niches within host cells. However, the Brucella T4SS effectors and their functions have not been fully identified. In this study, we investigated the function of Brucella RS15060, a novel T4SS effector discovered in our previous study, on the bacterial biological characteristics and pathogenesis by construction of the gene deletion and complementation strains. We found that deletion of the rs15060 gene weakened abilities of Brucella to replicate within host cells and establish chronic infection in mice but enhanced abilities to adhere/invade HeLa cells and evade lysosomal degradation in the early stage of infection. In addition, the rs15060 deletion Brucella strain showed significant changes in bacterial shape, cell wall thickness, and sensitivity to bactericidal factors. Furthermore, the rs15060 deletion strain showed an increased synthesis of bacterial lipopolysaccharide core and induced a stronger host's inflammatory response. The Brucella rs15060 complementation strain restored the altered biological characteristics. Moreover, BLASTP prediction and 3D structure simulation revealed that the Brucella RS15060 contains NAD(P)-binding and active motifs in structure, which are important for proteins to exert NAD dependent epimerase/dehydratase activity. The complementation strain with mutation on NAD(P)-binding and/or active motifs of RS15060 did not restore the altered characteristics, suggesting that the Brucella RS15060 is a potential NAD dependent epimerase/dehydratase, and the predicted NAD(P)-binding and/or active motifs play an important role on bacterial cell wall and LPS core synthesis, which is crucial for maintaining bacterial morphology and exerting virulence.

Bacteria commonly live in a spatially organized biofilm assemblage. The metabolic activity inside the biofilm leads to segmented physiological microenvironments. In nature, bacteria possess several pleomorphic forms to withstand certain ecological alterations. We hypothesized that pleomorphism also exists within the biofilm, which can be considered as the fundamental niche for bacteria. We report a distinct pattern of cell size variation throughout the biofilm of Bacillus subtilis. Cell size heterogeneity was observed in biofilm development, wherein the frequency of long cells is higher in outer regions, whereas lower in inner regions. Moreover, compared to planktonic cells, bacteria in the biofilm mode reduce their geometric ratio from 8.34 to 3.69 and 2.65 in the outer and inner regions, respectively. There were no significant differences observed in nutrient diffusion from the outer to the inner region, and more than 73 % of cells in the inner region were viable. However, the inner and middle regions were more acidic than the outer of the biofilm. Conclusively, growth rate-independent cell size reduction at low pH suggests that the resulting phenotype switching within biofilm was observed due to the pH gradient of neutral to acidic from the outer to the core of the biofilm. This gradient of H⁺ ions concentration may create exploitative stress within the biofilm, which could favor specific pleomorphic cells to thrive in their specialized niches. By understanding the cell size variation in response to the local environment, we propose a model of biofilm formation by pleomorphic cells.

H-NS is a prokaryotic histone-like protein that binds to bacterial chromosomal DNA with important regulatory roles in gene expression. Unlike histone proteins, hitherto post-translational modifications of H-NS are still largely uncharacterized, especially in bacterial pathogens. Salmonella Typhimurium is a primary enteric pathogen and its virulence is mainly dependent on specialized type III secretion systems (T3SSs), which were evolutionarily acquired via horizontal gene transfer. Previous studies have shown that H-NS plays a critical role in silencing foreign T3SS genes. Here, we found that H-NS is phosphorylated at multiple residues in S. Typhimurium, including S45, Y61, S78, S84, T86, and T106. Notably, we demonstrated that phosphorylation of H-NS S78 promotes its dissociation from DNA via a mechanism dependent on dimer formation, thereby leading to transcriptional activation of target genes. Functionally, phosphoryl-H-NS contributes to the expression of T3SS-associated proteins and hence increases bacterial virulence during infection. Therefore, our study reveals a novel mechanism by which covalent modifications of prokaryotic histone-like proteins regulate bacterial virulence of an important human pathogen.

Salmonella is a foodborne pathogen that predominantly resides in the intestinal tract of humans and animals. Infections caused by Salmonella can lead to various illnesses, including gastroenteritis, bacteremia, septicemia, and focal infections, with severe cases potentially resulting in host mortality. The mechanisms by which Salmonella invades host cells and disseminates throughout the body are partly understood, but there are still many scientific questions to be solved. This review aims to synthesize existing research on the interactions between Salmonella and hosts, detailing a comprehensive infection mechanism from adhesion and invasion to intracellular propagation and systemic spread. Overuse of antibiotics contributes to the emergence of drug-resistant Salmonella strains. An in-depth analysis of the mechanism of Salmonella infection will provide a theoretical basis for the development of novel Salmonella control strategies. These innovative control strategies include antibiotic adjuvants, small molecules, phages, attenuated vaccines, and probiotic therapies, which show huge potential in controlling Salmonella infection.

Light-controlled motility is advantageous for photosynthetic prokaryotes to better survive in environment with constantly changing light conditions. For cyanobacteria, light is both an energy source for photosynthesis and a stress factor. Consequently, some cyanobacteria evolved the ability to control type-IV pili (T4P)-mediated surface motility using a chemotaxis-like system in response to light signals. Extensive studies on the mechanism of phototaxis has been conducted in the spherical Synechocystis sp. PCC 6803 and the filamentous strain Nostoc punctiforme, while less is explored in rod-shaped cyanobacteria such as Synechococcus species. In this study, we investigated the phototaxis pathway in the unicellular rod-shaped cyanobacterium Synechococcus elongatus UTEX 3055, which exhibits bidirectional phototaxis using a single tax1 operon, in contrast to more complex and multiple gene clusters revealed in Synechocystis sp. PCC 6803. Results obtained by protein-protein interaction assays and protein subcellular localization experiments indicated that proteins encoded by the tax1 operon form large clusters that asymmetrically distributed both between the two poles and within the same pole. In vitro phosphorylation assays and site-directed mutations of conserved phosphorylation sites in PixL_Se, PixG_Se and PixH_Se demonstrate that PixL_Se acts as a histidine kinase, and PixG_Se and PixH_Se as response regulators for signal transduction. We further show that PixG_Se and PixH_Se are recruited to cell poles via interactions with the N-terminal region of PixL_Se. While phosphotransfer reactions in this signaling pathway are critical for phototactic signaling, the two response regulators appear to play different roles in the control of phototaxis. This study provides a framework for further investigation into the complex phototactic signaling network in rod-shaped cyanobacteria with clearly defined cell poles in contrast to round shaped Synechocystis species with virtual cells poles through light-lensing effect.

Xylella fastidiosa subsp. pauca (Xfp) currently presents a serious threat to agriculture in Europe and in the Mediterranean, following its discovery in several countries. Addressing this bacterial plant disease with traditional agricultural practices and management strategies has proven inadequate, highlighting the urgent need for effective and environmentally safe antibacterial solutions. In this study, we explored the antibacterial activity of the lactic acid bacterium Leuconostoc mesenteroides strain MS4-derived bacteriocins against Xfp, utilizing a combination of in vitro and in planta experiments. In particular, the cell-free precipitate (CFP) derived from strain MS4 culture in MRS broth, suppressed Xfp growth on BCYE agar plate, whereas protease K-treated CFP was inactive, highlighting the presence of antimicrobial compounds of proteinaceous nature. Additionally, fluorescence and transmission electron microscopy analyses showed that the CFP exhibits a bactericidal effect on Xfp cells, characterized by membrane disruption and subsequent cellular damage. The whole-genome sequencing and bioinformatic analysis revealed that MS4 genome consists of a circular chromosome of 1860,891 bp and a circular plasmid of 37,317 bp and most importantly to encompass six bacteriocin-encoding genes, with a peptide size ranging from 45 to 59 amino acids. MALDI-TOF/TOF MS and RPLC-ESI-MS assays performed on cell-free supernatant (CFS) confirmed the secretion of four (out of 6) bacteriocins (denoted MK-45, MR-53, MW-56, and MG-58) by MS4 in MRS broth. In spot assays, these bacteriocins displayed significant lethality against Xfp, with a minimum lethal concentration between 0.2 and 0.4 mg/mL. The application of CFP on Xfp-infected Nicotiana benthamiana plants, implemented both as preventive and curative approach, successfully controlled the infection, resulting in no visible symptoms 40 days post-inoculation. The finding of MS4 as a natural source of various potent bacteriocins against Xfp, coupled with a significant production under low-cost and uncomplicated laboratory conditions, make of MS4 a cost-effective and realistic option for sustainable management of Xf-related diseases.

The rise of antimicrobial resistance as a global health concern has led to a strong interest in compounds able to inhibit the growth of bacteria without detectable levels of resistance evolution. A number of these compounds have been reported in recent years, including the tridecaptins, a small family of lipopeptides typified by the synthetic analogue octyl-tridecaptin A₁. Hypothesizing that prior reports of negligible resistance evolution have been due in part to limitations in the laboratory evolution systems used, we have attempted to select for resistant mutants using a soft agar gradient evolution (SAGE) system developed by our lab. Following optimization of the media conditions by incorporation of the anti-synaeresis agent xanthan gum into the agar matrix, we successfully evolved high-level resistance to both octyl-tridecaptin A₁ as well as the challenging lipopeptide antibiotic polymyxin B. Decreased tridecaptin susceptibility was linked to mutations in outer membrane proteins ompC, lptD and mlaA, with the effect of these genes confirmed through a mix of allelic replacement and knockout studies. Overall, this work demonstrates the robust evolutionary potential of bacteria, even in the face of challenging antimicrobial agents.

The spread of multidrug-resistant (MDR) bacteria and their resistance genes along the food chain and the environment has become a global threat aggravated by incorrect disinfection strategies. This study analysed the effect of induction by sub-inhibitory concentrations of eugenol - a major ingredient in clove essential oil commonly used in disinfectant agents - on the phenotypic and genotypic response of MDR Enterococcus faecalis E9.8 strain, selected based on the phenotypic response of other enterococci. Eugenol treatment irreversibly reduced several antibiotics' minimum inhibitory concentration (MIC), confirmed by kinetic studies for kanamycin, erythromycin, and tetracycline. Furthermore, transcriptomic analysis indicated the reversion of antibiotic resistance through direct and indirect measures, such as down-regulation of genes coding for proteins involved in antibiotic resistance, toxin resistance and virulence factors. Regarding antibiotic resistance genes (ARGs), ten differentially expressed genes (five down-regulated and five up-regulated genes) were related to the main transporter families, which present key targets in antibiotic resistance reversion. Our study thus highlights the importance of considering indirectly related genes as targets for antibiotic resistance reversion besides ARGs sensu stricto. These results allow us to propose using eugenol as an antibiotic resistance reversing agent to be included in disinfectant solutions as an excellent alternative to limit the spread of MDR bacteria and their ARGs in the food chain and the environment.

Pseudomonas aeruginosa is a prominent respiratory pathogen in cystic fibrosis (CF) patients, thriving in the hypoxic airway mucus. Previous studies have established the role of the oxygen-binding hemerythrin, Mhr, in enhancing P. aeruginosa's fitness under microaerobic conditions. However, the specific mechanisms by which Mhr operates remain unclear. This study uniquely identifies Mhr as an effector of the H2-Type VI Secretion System (H2-T6SS) and elucidates its role in the transport and interaction mechanisms that confer a growth advantage under microaerobic conditions. Our findings demonstrate that mhr expression is directly regulated by Anr and Dnr. Western blot analysis confirms that Mhr is secreted extracellularly via the H2-T6SS. The oxygen-binding Mhr re-enters P. aeruginosa through the OprG porin. Then, Mhr interacts with cbb3-type cytochrome c oxidase (cbb3-CcO) subunits CcoP1/CcoP2, significantly impacting intracellular NADH/NAD⁺ levels. These insights suggest that the T6SS-mediated secretion and transport of Mhr represent a novel mechanism by which P. aeruginosa acquires and delivers oxygen, potentially enhancing microaerobic respiration, energy production, and growth under microaerobic conditions.