The (p)ppGpp synthetase Rsh promotes rifampicin tolerant persister cell formation in Brucella abortus by regulating the type II toxin-antitoxin module mbcTA.

Persister cells are transiently tolerant to antibiotics and are associated with recalcitrant chronic infections due to recolonization of host cells after antibiotic removal. Brucella spp. are facultative pathogens that establish intracellular infection cycles in host cells which results in chronic persistent infections. Brucella abortus forms multi-drug persister cells which are promoted by the (p)ppGpp synthetase Rsh during rifampicin exposure. Here, we confirmed that Rsh promoted persister cells formation in B. abortus stationary phase treated with rifampicin and enrofloxacin. Deletion of the gene for Rsh decreased persister cells level in the presence of these drugs in different growth phases. However, persister cells formation by deletion strain varied in different growth phases in the presence of other antibiotics. Rsh also was involved in persister cells formation during rifampicin treatment under certain stress conditions, including acidic conditions, exposure to PBS, and heat stress. Moreover, Rsh impacted persister cell levels during rifampicin or enrofloxacin treatment in RAW264.7 macrophages. Certain typeIItoxin-antitoxin modules were upregulated under various stress conditions in B. abortus. We established that Rsh positively regulated the type II toxin-antitoxin mbcTA. Moreover, rifampicin-tolerant persister cells formation was elevated and ATP levels were decreased when mbcTA promoter was overexpressed in Rsh deletion background in stationary phase. Our results establish that (p)ppGpp synthetase Rsh plays a key role in B. abortus persistence and may serve as a potent novel target in combination with rifampicin in the development of new therapeutic approaches and prevention strategies to treat chronic infections of Brucella.

A potential virulence factor: Brucella flagellin FliK does not affect the main biological properties but inhibits the inflammatory response in RAW264.7 cells.

The bacterial flagellum is an elongated filament that protrudes from the cell and is responsible for bacterial motility. It can also be a pathogen-associated molecular pattern (PAMP) that regulates the host immune response and is involved in bacterial pathogenicity. In contrast to motile bacteria, the Brucella flagellum does not serve a motile purpose. Instead, it plays a role in regulating Brucella virulence and the host's immune response, similar to other non-motile bacteria. The flagellin protein, FliK, plays a key role in assembly of the flagellum and also as a potential virulence factor involved in the regulation of bacterial virulence and pathogenicity. In this study, we generated a Brucella suis S2 flik gene deletion strain and its complemented strain and found that deletion of the flik gene has no significant effect on the main biological properties of Brucella, but significantly enhanced the inflammatory response induced by Brucella infection of RAW264.7 macrophages. Further experiments demonstrated that the FliK protein was able to inhibit LPS-induced cellular inflammatory responses by down-regulating the expression of MyD88 and NF-κB, and by decreasing p65 phosphorylation in the NF-κB pathway; it also inhibited the expression of NLRP3 and caspase-1 in the NLRP3 inflammasome pathway. In conclusion, our study suggests that Brucella FliK may act as a virulence factor involved in the regulation of Brucella pathogenicity and modulation of the host immune response.

Regulation of the Gene for Alanine Racemase Modulates Amino Acid Metabolism with Consequent Alterations in Cell Wall Properties and Adhesive Capability in Brucella spp.

Brucella, a zoonotic facultative intracellular pathogenic bacterium, poses a significant threat both to human health and to the development of the livestock industry. Alanine racemase (Alr), the enzyme responsible for alanine racemization, plays a pivotal role in regulating virulence in this bacterium. Moreover, Brucella mutants with alr gene deletions (Δalr) exhibit potential as vaccine candidates. However, the mechanisms that underlie the detrimental effects of alr knockouts on Brucella pathogenicity remain elusive. Here, initially, we conducted a bioinformatics analysis of Alr, which demonstrated a high degree of conservation of the protein within Brucella spp. Subsequent metabolomics studies unveiled alterations in amino acid pathways following deletion of the alr gene. Furthermore, alr deletion in Brucella suis S2 induced decreased resistance to stress, antibiotics, and other factors. Transmission electron microscopy of simulated macrophage intracellular infection revealed damage to the cell wall in the Δalr strain, whereas propidium iodide staining and alkaline phosphatase and lactate dehydrogenase assays demonstrated alterations in cell membrane permeability. Changes in cell wall properties were revealed by measurements of cell surface hydrophobicity and zeta potential. Finally, the diminished adhesion capacity of the Δalr strain was shown by immunofluorescence and bacterial enumeration assays. In summary, our findings indicate that the alr gene that regulates amino acid metabolism in Brucella influences the properties of the cell wall, which modulates bacterial adherence capability. This study is the first demonstration that Alr impacts virulence by modulating bacterial metabolism, thereby providing novel insights into the pathogenic mechanisms of Brucella spp.

(P)ppGpp synthetase Rsh participates in rifampicin tolerance of persister cells in Brucella abortus in vitro.

Brucella abortus is facultative intracellular pathogen that causes chronic persistent infections and results in abortion and infertility in food animals. Recurrent infections can be one of the results of persister cells formation that transiently displays phenotypic tolerance to high dose of antibiotics treatment. We examined persister cells formation of B. abortus strain A19 in stationary phase and investigated a potential role for the (p)ppGpp synthetase Rsh in this process. We found that B. abortus stationary phase cells can produce higher levels of multi-drugs tolerant persister cells in vitro under high dose of antibiotics (20 × MIC) exposure than do exponential phase cells. Persister cell formation was also induced with environmental stressors pH 4.5, 0.01 M PBS (pH7.0), 2% NaCl and 25 °C, upon exposure to ampicillin, enrofloxacin and rifampicin. Persister cells were not formed following exposure to 1 mM H2O2. The numbers of persister cells were significantly increased following uptake of B. abortus stationary phase cells by RAW264.7 macrophages in contrast with cultures in TSB liquid medium. Environmental stressors to B. abortus significantly increased expression of rsh mRNA level. The rsh null mutant (Δrsh) formed significantly fewer persister cells than the complemented (CΔrsh) and wildtype (WT) strains under high dose of rifampicin in vitro. These data for the first time demonstrate that B. abortus can produce multi-drug tolerant persister cells in stationary phase. The (p)ppGpp synthetase Rsh is necessary for persister cell formation in B. abortus in the presence of rifampicin. On this basis, a new understanding of the recurrent infections of Brucella was advanced, thus provided a new basis for revelation of pathogenic mechanism of the chronic persistent infection in Brucella.

Alr Gene in Brucella suis S2: Its Role in Lipopolysaccharide Biosynthesis and Bacterial Virulence in RAW264.7.

Brucella suis, the causative agent of brucellosis, poses a significant public health and animal husbandry threat. However, the role of the alanine racemase (alr) gene, which encodes alanine racemase in Brucella, remains unclear. Here, we analyzed an alr deletion mutant and a complemented strain of Brucella suis S2. The knockout strain displayed an unaltered, smooth phenotype in acriflavine agglutination tests but lacked the core polysaccharide portion of lipopolysaccharide (LPS). Genes involved in the LPS synthesis were significantly upregulated in the deletion mutant. The alr deletion strain exhibited reduced intracellular viability in the macrophages, increased macrophage-mediated killing, and upregulation of the apoptosis markers. Bcl2, an anti-apoptotic protein, was downregulated, while the pro-apoptotic proteins, Bax, Caspase-9, and Caspase-3, were upregulated in the macrophages infected with the deletion strain. The infected macrophages showed increased mitochondrial membrane permeability, Cytochrome C release, and reactive oxygen species, activating the mitochondrial apoptosis pathway. These findings revealed that alanine racemase was dispensable in B. suis S2 but influenced the strain's rough features and triggered the mitochondrial apoptosis pathway during macrophage invasion. The deletion of the alr gene reduced the intracellular survival and virulence. This study enhances our understanding of the molecular mechanism underlying Brucella's survival and virulence and, specifically, how alr gene affects host immune evasion by regulating bacterial LPS biosynthesis.

Brucella BtpB Manipulates Apoptosis and Autophagic Flux in RAW264.7 Cells.

Brucella transfers effectors into host cells, manipulating cellular processes to its advantage; however, the mechanism by which effectors regulate cellular processes during infection is poorly understood. A growing number of studies have shown that apoptosis and autophagy are critical mechanisms for target cells to cope with pathogens and maintain cellular homeostasis. BtpB is a Brucella type IV secretion system effector with a complex mechanism for manipulating host infection. Here, we show that the ectopic expression of BtpB promoted DNA fragmentation. In contrast, an isogenic mutant strain, ΔbtpB, inhibited apoptosis compared to the wild-type strain B. suis S2 in RAW264.7 cells. In addition, BtpB inhibited autophagy, as determined by LC3-II protein levels, the number of LC3 puncta, and p62 degradation. We also found that BtpB reduced autophagolysosome formation and blocked the complete autophagic flux. Moreover, our results revealed that the autophagy inhibitor, chloroquine, reduces Brucella's intracellular survival. Overall, our data unveil new mechanisms of virulence implicating the effector BtpB in regulating host intracellular infection.

An ArsR Transcriptional Regulator Facilitates Brucella sp. Survival via Regulating Self and Outer Membrane Protein.

The arsenic acid-resistant (ArsR) family transcriptional regulators are widely distributed in microorganisms, including in the facultative intracellular pathogen Brucella spp. ArsR proteins are implicated in numerous biological processes. However, the specific roles of ArsR family members in Brucella remain obscure. Here, we show that ArsR6 (BSS2_RS07325) is required for Brucella survival both under heat, oxidative, and osmotic stress and in a murine infection model in vivo. RNA-seq and ChIP-seq reveal that 34 potential target genes for ArsR6 protein were identified, among which eight genes were up-regulated and 26 genes were down-regulated, including outer membrane protein 25D (Omp25D). ArsR6 autoregulates its own expression to maintain bacterial intracellular Cu/Ni homeostasis to benefit bacterial survival in hostile environments. Moreover, ArsR6 also regulates the production of virulence factor Omp25D, which is important for the survival of Brucella under stress conditions. Significantly, Omp25D deletion strain attenuated in a murine infection model in vivo. Altogether, our findings reveal a unique mechanism in which the ArsR family member ArsR6 autoregulates its expression and also modulates Omp25D expression to maintain metal ion homeostasis and virulence in Brucella.

Protective effect of recombinant Lactobacillus plantarum against H2O2-induced oxidative stress in HUVEC cells.

This study probed the protective effect of recombinant Lactobacillus plantarum against hydrogen peroxide (H2O2)-induced oxidative stress in human umbilical vein endothelial cells (HUVECs). We constructed a new functional L. plantarum (NC8-pSIP409-alr-angiotensin-converting enzyme inhibitory peptide (ACEIP)) with a double-gene-labeled non-resistant screen as an expression vector. A 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay was carried out to determine the cell viability of HUVEC cells following pretreatment with NC8-pSIP409-alr-ACEIP. Flow cytometry (FCM) was used to determine the apoptosis rate of HUVEC cells. Cysteinyl aspartate specific proteinase (caspase)-3/8/9 activity was also assayed and western blotting was used to determine protein expression of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), inducible nitric oxide synthase (iNOS), nicotinamide adenine dinucleotide phosphate oxidase 2 (gp91phox), angiotensin II (AngII), and angiotensin-converting enzyme 2 (ACE2), as well as corresponding indicators of oxidative stress, such as reactive oxygen species (ROS), mitochondrial membrane potential (MMP), malondialdehyde (MDA), and superoxide dismutase (SOD). NC8-pSIP409-alr-ACEIP attenuated H2O2-induced cell death, as determined by the MTT assay. NC8-pSIP409-alr-ACEIP reduced apoptosis of HUVEC cells by FCM. In addition, compared to the positive control, the oxidative stress index of the H2O2-induced HUVEC (Hy-HUVEC), which was pretreated by NC8-pSIP409-alr-ACEIP, iNOS, gp91phox, MDA, and ROS, was decreased obviously; SOD expression level was increased; caspase-3 or -9 was decreased, but caspase-8 did not change; Bcl-2/Bax ratio was increased; permeability changes of mitochondria were inhibited; and loss of transmembrane potential was prevented. Expression of the hypertension-related protein (AngII protein) in HUVEC cells protected by NC8-pSIP409-alr-ACEIP decreased and expression of ACE2 protein increased. These plantarum results suggested that NC8-pSIP409-alr-ACEIP protects against H2O2-induced injury in HUVEC cells. The mechanism for this effect is related to enhancement of antioxidant capacity and apoptosis.

Study of the rational dose of propofol in elderly patients under bispectral index monitoring during total intravenous anesthesia: A PRISMA-compliant systematic review.

BACKGROUND: Propofol has been used widely as an anesthetic for elderly patients; however, the drug instructions only indicate that the need for maintenance of general anesthesia in elderly patients is reduced, and not the extent of the reduction. This study has summarized the usage of propofol in total intravenous anesthesia under bispectral index (BIS) monitoring and determined the optimum dosage of propofol for elderly patients. METHODS: The study comprised 156 patients undergoing elective surgery under general anesthesia divided into 2 groups according to their age: the elderly group (O group) and nonelderly group (Y group). BIS monitoring was used in both groups during the operation, and propofol and remifentanil were used to maintain anesthesia. The preoperative special conditions, intraoperative maintenance of propofol, remifentanil, fentanyl, cis-atracurium, vasoactive drug use, and hemodynamic changes were summarized. RESULTS: Propofol maintenance in the O group was 3.372 ±â€Š0.774 mg/(kg h), which was significantly lesser than that in Y group (P < 0.05). The incidence of cardiovascular and cerebrovascular diseases and the use rate of vasoactive drugs in the O group were significantly higher than in the Y group (P < 0.05). CONCLUSION: Propofol maintenance in the O group was significantly lower than that in the nonelderly group; this indicates that the anesthetic drug delivery rate for elderly patients should be reduced.