Genetic distribution, characterization, and function of Escherichia coli type III secretion system 2 (ETT2).

Many Gram-negative bacteria use type Ⅲ secretion system (T3SS) to inject effector proteins and subvert host signaling pathways, facilitating the growth, survival, and virulence. Notably, some bacteria harbor multiple distinct T3SSs with different functions. An extraordinary T3SS, the Escherichia coli Type III Secretion System 2 (ETT2), is widespread among Escherichia coli (E. coli) strains. Since many ETT2 carry genetic mutations or deletions, it is thought to be nonfunctional. However, increasing studies highlight ETT2 contributes to E. coli pathogenesis. Here, we present a comprehensive overview of genetic distribution and characterization of ETT2. Subsequently, we outline its functional potential, contending that an intact ETT2 may retain the capacity to translocate effector proteins and manipulate the host's innate immune response. Given the potential zoonotic implications associated with ETT2-carrying bacteria, further investigations into the structure, function and regulation of ETT2 are imperative for comprehensive understanding of E. coli pathogenicity and the development of effective control strategies.

The aroA and luxS Double-Gene Mutant Strain Has Potential to Be a Live Attenuated Vaccine against Salmonella Typhimurium.

Salmonella Typhimurium (S. Typhimurium) is a zoonotic pathogen posing a threat to animal husbandry and public health. Due to the emergence of antibiotic-resistant strains, alternative prevention and control strategies are needed. Live attenuated vaccines are an ideal option that provide protection against an S. Typhimurium pandemic. To develop a safe and effective vaccine, double-gene mutations are recommended to attenuate virulence. In this study, we chose aroA and luxS genes, whose deletion significantly attenuates S. Typhimurium's virulence and enhances immunogenicity, to construct the double-gene mutant vaccine strain SAT52ΔaroAΔluxS. The results show that the mutant strain's growth rate, adherence and invasion of susceptible cells are comparable to a wild-type strain, but the intracellular survival, virulence and host persistence are significantly attenuated. Immunization assay showed that 106 colony-forming units (CFUs) of SAT52ΔaroAΔluxS conferred 100% protection against wild-type challenges; the bacteria persistence in liver and spleen were significantly reduced, and no obvious pathological lesions were observed. Therefore, the double-gene mutant strain SAT52ΔaroAΔluxS exhibits potential as a live attenuated vaccine candidate against S. Typhimurium infection.

The functions of EF-hand proteins from host and zoonotic pathogens.

EF-hand proteins not only regulate biological processes, but also influence immunity and infection. In this review, we summarize EF-hand proteins' functions in host and zoonotic pathogens, with details in structures, Ca2+ affinity, downstream targets and functional mechanisms. Studies entitled as EF-hand-related but with less solid features were also discussed. We believe it could raise cautions and facilitate proper research strategy for researchers.

A lytic phage to control multidrug-resistant avian pathogenic Escherichia coli (APEC) infection.

The inappropriate use of antibiotics has led to the emergence of multidrug-resistant strains. Bacteriophages (phages) have gained renewed attention as promising alternatives or supplements to antibiotics. In this study, a lytic avian pathogenic Escherichia coli (APEC) phage designated as PEC9 was isolated and purified from chicken farm feces samples. The morphology, genomic information, optimal multiplicity of infection (MOI), one-step growth curve, thermal stability, pH stability, in vitro antibacterial ability and biofilm formation inhibition ability of the phage were determined. Subsequently, the therapeutic effects of the phages were investigated in the mice model. The results showed that PEC9 was a member of the siphovirus-like by electron microscopy observation. Biological characterization revealed that it could lyse two serotypes of E. coli, including O1 (9/20) and O2 (6/20). The optimal multiplicity of infection (MOI) of phage PEC9 was 0.1. Phage PEC9 had a latent period of 20 min and a burst period of 40 min, with an average burst size of 68 plaque-forming units (PFUs)/cell. It maintained good lytic activity at pH 3-11 and 4-50°C and could efficiently inhibit the bacterial planktonic cell growth and biofilm formation, and reduce bacterial counts within the biofilm, when the MOI was 0.01, 0.1, and 1, respectively. Whole-genome sequencing showed that PEC9 was a dsDNA virus with a genome of 44379 bp and GC content of 54.39%. The genome contains 56 putative ORFs and no toxin, virulence, or resistance-related genes were detected. Phylogenetic tree analysis showed that PEC9 is closely related to E. coli phages vB_EcoS_Zar3M, vB_EcoS_PTXU06, SECphi18, ZCEC10, and ZCEC11, but most of these phages exhibit different gene arrangement. The phage PEC9 could successfully protect mice against APEC infection, including improved survival rate, reduced bacterial loads, and organ lesions. To conclude, our results suggest that phage PEC9 may be a promising candidate that can be used as an alternative to antibiotics in the control of APEC infection.

Plasma levels of 12 different cytokines correlate to PD-1 inhibitor combined chemotherapy responses in advanced non-small-cell lung cancer patient.

BACKGROUND: Targeted anti-programmed death receptor 1 (PD-1) monoclonal antibodies, when combined with chemotherapy, have shown improved outcomes in non-small cell lung cancer (NSCLC). However, it is important to note that not all patients benefit from this treatment, and there is a pressing need for more reliable efficacy measures and potential predictors of outcome. Cytokines, which are important molecules in the immune system, have been considered as potential biomarkers in clinical settings, but their precise clinical use remains unclear. In this study, our objective was to assess whether the levels of cytokines in the patient's blood sample are associated with tumor response to anti-PD-1 monoclonal antibodies combined with chemotherapy as well as the survival of patients with advanced non-small cell lung cancer. MATERIALS AND METHODS: A total of 12 plasma cytokines were measured in advanced NSCLC patients (n = 35) and healthy individuals (n = 26) using multi-microsphere flow immunofluorescence. The relationship between cytokine levels and clinical response was analyzed using nonparametric Wilcoxon matched-pair ranked tests. Progression-free survival (PFS) time was recorded for all patients through radiographic outcome assessment and telephone follow-up. Survival curves were generated using the Kaplan-Meier and log-rank tests, and the thresholds for cytokines were determined using receiver operating characteristic analysis (ROC). RESULTS: The expression levels of interleukin IL-6, IL-1 ß, IFN-γ, IL-12p70, and TNF-α were significantly lower in the control group than those in the NSCLC group (p = 0.001, p = 0.0028, p = 0.019, p = 0.0001, p = 0.0021). High IL-10 levels at baseline and after 4 cycles of treatment conferred a worse prognosis; in addition, high TNF-α levels in patients after two cycles of immunochemotherapy suggested drug resistance. High levels of IL-6 and IFN-γ in patients undergoing four cycles of immunochemotherapy were associated with worse PFS. CONCLUSIONS: Our study suggests that cytokines can serve as detection indicators for predicting efficacy in non-small cell lung cancer patients undergoing anti-PD-1 combined with chemotherapy treatment. Elevated levels of IL-10, TNF-α, IL-6, and IFN-γ in the plasma may indicate a higher likelihood of experiencing a worse clinical outcome.

Association between serum levels of 12 different cytokines and short-term efficacy of anti-PD-1 monoclonal antibody combined with chemotherapy in advanced gastric cancer.

BACKGROUND: Gastric cancer (GC) is characterized by aggressive tumor growth and poor prognosis. The benefits of targeted anti-programmed death receptor 1 (PD-1) monoclonal antibody combined with chemotherapy have not yet been characterized. The tumor microenvironment and circulating factors have garnered interest as possible predictors of response and prognosis. The aim of this study was to evaluate whether cytokine levels in the serum of patients were related to tumor response to anti-PD-1 monoclonal antibody combined with chemotherapy and survival in advanced GC. MATERIALS AND METHODS: Preoperative serum samples were collected from patients with GC (n = 52) and healthy individuals (n = 31). The levels of 12 different cytokines were measured using a multiple microsphere flow immunofluorescence assay. The association between cytokine levels and clinical response was analyzed using nonparametric Wilcoxon matched-pair ranked tests. Progression-free survival (PFS) time for all patients was recorded via evaluation of imaging results and follow-up via telephone. Kaplan-Meier and log-rank tests were used to plot survival curves. RESULTS: The levels of interleukin (IL)-6, IL-1ß, interferon (IFN)-γ, IL-17, and IL-12p70 in the control group were significantly lower than those in the GC group (p = 0.0002, p = 0.0065, p = 0.0003, p = 0.0303, and p = 0.0295, respectively). The level of IL-4 was significantly higher in healthy individuals than that in patients with GC (p = 0.0201). The cytokine levels in the good responder group were higher than those in the poor responder group before therapy. Patients treated with immunochemotherapy showed an overall reduction in all cytokine levels after treatment initiation. A high baseline level of IFN-γ was associated with a better prognosis. However, high IL-6 levels in patients after two cycles of immunochemotherapy indicated resistance. High IL-4 levels in patients treated with four cycles of immunochemotherapy were associated with better PFS. CONCLUSIONS: Our study suggests that low levels of IFN-γ before immune checkpoint inhibitor treatment may be useful for the detection of a poor immunological status. Hence, a reduction in IL-6 levels is predictive of a longer PFS, and increased IL-4 levels are predictive of a good response. IL-4 and IL-6 may, therefore, serve as promising circulating predictive biomarkers for patients who can benefit from anti-PD-1 monoclonal antibodies administered in combination with chemotherapy.

Preliminary Study on the In Vitro Antitumor Effects of Nidus Vespae on Gastric Cancer.

OBJECTIVE: The aim of this study was to investigate the in vitro antitumor effects of Nidus Vespae on gastric cancer and its ability to promote immune function. METHODS: Cell viability was detected by the Cell Counting Kit-8 (CCK-8) assay. Cell cycle distribution and apoptosis were detected using flow cytometry. The THP-1 human monocytic cell line was used as a source of monocytic effector cells for analyzing proliferation and dendritic cell (DC) induction. Enzyme-linked immunosorbent assay was used to detect cytokine production, and multicolor flow cytometry was used to study the phenotype and functionality of THP-1 DCs. RESULTS: A high concentration (>10 mg/mL) of Nidus Vespae decoction (NVD) inhibited SGC-7901 gastric cancer cell growth by inducing G2/M cell cycle arrest and apoptosis. However, a low concentration (≤10 mg/mL) of NVD significantly increased the proliferative ability of THP-1 in serum-containing medium and caused an increase in dendritic protrusions with the typical morphology of DCs compared to the negative control in serum-free medium. The THP-1 DCs had significantly increased expression of cluster of differentiation 11c (CD11c), CD40, CD80, CD83, and CD86, as well as secretion of tumor necrosis factor-alpha. Furthermore, the supernatant of THP-1 DCs significantly inhibited the proliferation of gastric cancer cells by inducing apoptosis and G1/S cell cycle arrest. CONCLUSIONS: Our findings suggest that NVD not only directly inhibits the growth of gastric cancer cells but also exerts indirect antitumor effects by enhancing immune function. These results provide an important theoretical basis for the clinical application of Nidus Vespae in gastric cancer treatment.

A Small Protein but with Diverse Roles: A Review of EsxA in Mycobacterium-Host Interaction.

As a major effector of the ESX-1 secretion system, EsxA is essential for the virulence of pathogenic mycobacteria, such as Mycobacterium tuberculosis (Mtb) and Mycobacterium marinum (Mm). EsxA possesses an acidic pH-dependent membrane permeabilizing activity and plays an essential role by mediating mycobacterial escape from the phagosome and translocation to the cytosol for intracellular replication. Moreover, EsxA regulates host immune responses as a potent T-cell antigen and a strong immunoregulator. EsxA interacts with multiple cellular proteins and stimulates several signal pathways, such as necrosis, apoptosis, autophagy, and antigen presentation. Interestingly, there is a co-dependency in the expression and secretion of EsxA and other mycobacterial factors, which greatly increases the complexity of dissecting the precise roles of EsxA and other factors in mycobacterium-host interaction. In this review, we summarize the current understandings of the roles and functions of EsxA in mycobacterial infection and discuss the challenges and future directions.

Post-translational knockdown and post-secretional modification of EsxA determine contribution of EsxA membrane permeabilizing activity for mycobacterial intracellular survival.

Current genetic studies (e.g. gene knockout) have suggested that EsxA and EsxB function as secreted virulence factors that are essential for Mycobaterium tuberculosis (Mtb) intracellular survival, specifically in mediating phagosome rupture and translocation of Mtb to the cytosol of host cells, which further facilitates Mtb intracellular replicating and cell-to-cell spreading. The EsxA-mediated intracellular survival is presumably achieved by its pH-dependent membrane-permeabilizing activity (MPA). However, the data from other studies have generated a discrepancy regarding the role of EsxA MPA in mycobacterial intracellular survival, which has raised a concern that genetic manipulations, such as deletion of esxB-esxA operon or RD-1 locus, may affect other codependently secreted factors that could be also directly involved cytosolic translocation, or stimulate extended disturbance on other genes' expression. To avoid the drawbacks of gene knockout, we first engineered a Mycobacterium marinum (Mm) strain, in which a DAS4+ tag was fused to the C-terminus of EsxB to allow inducible knockdown of EsxB (also EsxA) at the post-translational level. We also engineered an Mm strain by fusing a SpyTag (ST) to the C-terminus of EsxA, which allowed inhibition of EsxA-ST MPA at the post-secretional level through a covalent linkage to SpyCatcher-GFP. Both post-translational knockdown and functional inhibition of EsxA resulted in attenuation of Mm intracellular survival in lung epithelial cells or macrophages, which unambiguously confirms the direct role of EsxA MPA in mycobacterial intracellular survival.

The ESX-1 Virulence Factors Downregulate miR-147-3p in Mycobacterium marinum-Infected Macrophages.

As important virulence factors of Mycobacterium tuberculosis, EsxA and EsxB not only play a role in phagosome rupture and M. tuberculosis cytosolic translocation but also function as modulators of host immune responses by modulating numerous microRNAs (miRNAs). Recently, we have found that mycobacterial infection downregulated miR-148a-3p (now termed miR-148) in macrophages in an ESX-1-dependent manner. The upregulation of miR-148 reduced mycobacterial intracellular survival. Here, we investigated miR-147-3p (now termed miR-147), a negative regulator of inflammatory cytokines (e.g., interleukin-6 [IL-6] and IL-10), in mycobacterial infection. We infected murine RAW264.7 macrophages with Mycobacterium marinum, a surrogate model organism for M. tuberculosis, and found that the esxBA-knockout strain (M. marinum ΔesxBA) upregulated miR-147 to a level that was significantly higher than that induced by the M. marinum wild-type (WT) strain or by the M. marinum ΔesxBA complemented strain, M. marinum ΔesxBA/pesxBA, suggesting that the ESX-1 system (potentially EsxBA and/or other codependently secreted factors) is the negative regulator of miR-147. miR-147 was also downregulated by directly incubating the macrophages with the purified recombinant EsxA or EsxB protein or the EsxBA heterodimer, which further confirms the role of the EsxBA proteins in the downregulation of miR-147. The upregulation of miR-147 inhibited the production of IL-6 and IL-10 and significantly reduced M. marinum intracellular survival. Interestingly, inhibitors of either miR-147 or miR-148 reciprocally compromised the effects of the mimics of their counterparts on M. marinum intracellular survival. This suggests that miR-147 and miR-148 share converged downstream pathways in response to mycobacterial infection, which was supported by data indicating that miR-147 upregulation inhibits the Toll-like receptor 4/NF-κB pathway.

Morphology-based classification of mycobacteria-infected macrophages with convolutional neural network: reveal EsxA-induced morphologic changes indistinguishable by naked eyes.

EsxA is an essential virulence factor for Mycobacterium tuberculosis (Mtb) pathogenesis as well as an important biomarker for Mtb detection. In this study, we use light microscopy and deep learning-based image analysis to classify the morphologic changes of macrophages infected by Mycobacterium marinum (Mm), a surrogate model for Mtb. Macrophages were infected either with the mCherry-expressing Mm wild type strain (Mm(WT)), or a mutant strain with deletion of the esxA-esxB operon (Mm(ΔEsxA:B)). The mCherry serves as an infection marker to train the convolution neural network (CNN) and to validate the classification results. Data show that CNN can distinguish the Mm(WT)-infected cells from uninfected cells with an accuracy of 92.4% at 2 hours postinfection (hpi). However, the accuracy at 12 and 24 hpi is decreased to ∼75% and ∼83%, respectively, suggesting dynamic morphologic changes through different stages of infection. The accuracy of discriminating Mm(ΔEsxA:B)-infected cells from uninfected cells is lower than 80% at all time, which is consistent to attenuated virulence of Mm(ΔEsxA:B). Interestingly, CNN distinguishes Mm(WT)-infected cells from Mm(ΔEsxA:B)-infected cells with ∼90% accuracy, implicating EsxA induces unique morphologic changes in macrophages. Deconvolutional analysis successfully reconstructed the morphologic features used by CNN for classification, which are indistinguishable to naked eyes and distinct from intracellular mycobacteria. This study presents a deep learning-aided imaging analytical tool that can accurately detect virulent mycobacteria-infected macrophages by cellular morphologic changes. The observed morphologic changes induced by EsxA warrant further studies to fill the gap from molecular actions of bacterial virulence factors to cellular morphology.

Brucella infection regulates peroxiredoxin-5 protein expression to facilitate intracellular survival by reducing the production of nitric oxide and reactive oxygen species.

Peroxiredoxin-5 (Prdx5) is a multifunctional protein involved in oxidative stress, apoptosis and inflammatory responses. However, how Prdx5 functions during microbial infections is rarely reported. In this study, we demonstrate that Brucella infection increased Prdx5 expression to promote its intracellular growth in macrophages. Further study show that B. abortus infection promoted its intracellular growth by decreasing the production of nitric oxide and reactive oxygen species. In addition, the expression of Prdx5 was independent on live Brucella and the type IV secretion system of Brucella. Instead, its expression was regulated by the lipopolysaccharide of Brucella. Moreover, Brucella infection increased Prdx5 expression in primary macrophage and mice. Collectively, these findings demonstrate for the first time that Prdx5 promotes Brucella intracellular growth by decreasing the production of NO and ROS. This finding provides new insights into the evasive strategies of Brucella and will be useful for the development of novel effective therapeutic approaches to treat Brucella infections.

Mycobacterium marinum down-regulates miR-148a in macrophages in an EsxA-dependent manner.

As a key virulence factor of Mycobacterium tuberculosis, EsxA is not only involved in phagosome rupture, but also functions in stimulation of immune responses in macrophages. Here, we report thatmiR-148a is down-regulated in the macrophages infected with Mycobacterium marinum (Mm). Using the knockout strain Mm∆EsxA/B, recombinant EsxA, EsxB and EsxA/B heterodimer proteins, we provide evidence that down-regulation of miR-148ais dependent on EsxA, and up-regulation of miR-148a reduces Mm intracellular survival. Moreover, up-regulation of miR-148a down-regulates the pro-inflammatory cytokines (e.g. TNF-α and IL-1ß) and the TLR4-mediated NF-κB activation. Together, miR-148a may function as an anti-inflammation modulator in responses to mycobacterial infection. Regulation of miR-148a may provide a novel venue in development of therapies in tuberculosis.

Identification of novel genes essential for Brucella abortus to establish infection by signature-tagged mutagenesis.

Brucella is a facultative intracellular bacterium, causing brucellosis, an important zoonosis worldwide. Brucella has no classic virulence factors, thus virulence is dependent on invasion of host cells and subsequent intracellular replication. Identification of key genes involved in Brucella virulence is important to further elucidate its pathogenesis. In this study, signature-tagged mutagenesis was used to identify novel genes involved in B. abortus infection in a mouse model. In total 3600 mutants were obtained, of which 56 were identified as attenuated mutants. Furthermore, 53 genes were identified to be inactivated by transposon insertion, including 19 genes previously reported to be essential for Brucella virulence and 34 others that were newly identified in this study. These genes were catalogued into 16 functional classifications, except for three that were not cited in the Clusters of Orthologous Groups database. Bioinformatics analysis revealed that energy production and conversion, amino acid transport and metabolism, as well as inorganic ion transport and metabolism were predominant functional classifications, suggesting that genes involved in these functions were crucial for Brucella virulence. In addition, the function of the identified pyruvate carboxylase (pyc) gene in bacterial virulence was confirmed using an allelic replacement pyc mutant and a mouse model. These findings provide novel genetic information associated with Brucella infection.

Identification of a novel, small, conserved hypothetical protein involved in Brucella abortus virulence by modifying the expression of multiple genes.

Brucellosis is an important zoonotic disease worldwide, caused by Brucella spp., which are facultative intracellular bacteria with no classic virulence factors, as virulence is dependent on the ability to invade and replicate within host cells. In this study, we identified a novel gene bab_RS22045 that encodes a small highly conserved protein in Rhizobiales. To investigate the role of this gene, a deletion mutant and complement strain were constructed. Virulence testing showed that bab_RS22045 is necessary for Brucella virulence, and was designated as virulence-related hypothetical protein, VhpA. The results of a cell infection experiment showed that vhpA was not associated with Brucella adherence to and invasion of HeLa cells, or further intracellular survival within RAW264.7 cells. The results of sensitivity testing showed the vhpA mutant had similar sensitivity to hydrogen peroxide, polymyxin B, and sodium nitroprusside as the wild-type (WT) strain. Interestingly, RNA-seq analysis showed that deletion of the vhpA gene affected the expression patterns of multiple Brucella genes, and the main four up-regulated genes and five down-regulated genes were further confirmed using quantitative real-time PCR analysis. Subsequently, a series of over-expression strains were constructed, and virulence testing showed that over-expression of four up-regulated genes (bab_RS17930, bab_RS17925, bab_RS26460, and bab_RS30050) significantly reduced virulence of the WT strain, and over-expression of bab_RS18680 in the vhpA mutant partially restored virulence, suggesting that vhpA plays an important role in Brucella virulence by changing the expression patterns of multiple genes. Additionally, heterogeneous complementary analysis showed that the homologous vhpA genes of Sinorhizobium meliloti and Agrobacterium tumefaciens could not restore virulence of the vhpA mutant, although VhpA is a highly conserved protein in Rhizobiales. Overall, a novel, small, hypothetical gene was identified that is associated with B. abortus virulence, which highlights the roles of small encoding genes in Brucella virulence.

The putative amino acid ABC transporter substrate-binding protein AapJ2 is necessary for Brucella virulence at the early stage of infection in a mouse model.

Brucellosis is a zoonotic bacterial disease caused by Brucella spp. The virulence of these bacteria is dependent on their ability to invade and replicate within host cells. In a previous study, a putative gene bab_RS27735 encoding an amino acid ABC transporter substrate-binding protein homologous to AapJ protein was found to be involved in Brucella abortus virulence. In this study, we successfully constructed a bab_RS27735 deletion mutant, Δ27735. Compared with the wild-type strain, the lipopolysaccharide pattern of the mutant was not changed, but the growth ability was slightly defected in the exponential phase. In tolerance tests, sensitivity of the Δ27735 mutant to oxidative stress, bactericidal peptides or low pH was not different from that of the wild-type strain. Cell infection assay showed that the mutant was reduced survival within macrophages but could efficiently escape lysosome degradation. The results of a virulence test showed that the Δ27735 mutant was attenuated in a mouse model at the early stage of infection but recovered its virulence at the late stage of infection. Meanwhile, the development of splenomegaly and histopathological lesions was observed in mice infected with either the wild-type strain or the mutant. These results are in line with the release of IL-12p40 and TNF-α into the peripheral blood of infected mice. Besides, expression of diverse genes was up-regulated in the Δ27735 mutant, which may contribute to the reduced virulence of the mutant. These data elucidated that the bab_RS27735 gene is necessary for B. abortus virulence at the early stage of infection in a mouse model.

Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion.

Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS) and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant ΔrfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the molecular mechanisms associated with Brucella rough mutant-induced macrophage cytotoxicity.

Characterization of Brucella abortus mutant strain Δ22915, a potential vaccine candidate.

Brucellosis, caused by Brucella spp., is an important zoonosis worldwide. Vaccination is an effective strategy for protection against Brucella infection in livestock in developing countries and in wildlife in developed countries. However, current vaccine strains including S19 and RB51 are pathogenic to humans and pregnant animals, limiting their use. In this study, we constructed the Brucella abortus (B. abortus) S2308 mutant strain Δ22915, in which the putative lytic transglycosylase gene BAB_RS22915 was deleted. The biological properties of mutant strain Δ22915 were characterized and protection of mice against virulent S2308 challenge was evaluated. The mutant strain Δ22915 showed reduced survival within RAW264.7 cells and survival in vivo in mice. In addition, the mutant strain Δ22915 failed to escape fusion with lysosomes within host cells, and caused no observable pathological damage. RNA-seq analysis indicated that four genes associated with amino acid/nucleotide transport and metabolism were significantly upregulated in mutant strain Δ22915. Furthermore, inoculation of ∆22915 at 105 colony forming units induced effective host immune responses and long-term protection of BALB/c mice. Therefore, mutant strain ∆22915 could be used as a novel vaccine candidate in the future to protect animals against B. abortus infection.