Impaired functions of human monocyte-derived dendritic cells and induction of regulatory T cells by pathogenic Leptospira.

Leptospirosis is a global zoonosis caused by pathogenic Leptospira. The disease outcome is influenced by the interplay between innate and adaptive immune responses. Dendritic cells (DCs) play a crucial role in shaping the adaptive immune response. A recent study revealed that pathogenic Leptospira limited the activation of human monocyte-derived dendritic cells (MoDCs) compared to non-pathogenic Leptospira, but their impact on T-cell responses has not been investigated. Our study is the first to explore how viable pathogenic and non-pathogenic Leptospira affect the interaction between human MoDCs and T cells. We found that MoDCs infected with pathogenic leptospires (L. interrogans serovar Pomona and a clinical isolate, MoDCs-P) exhibited lower levels of CD80 and CD83 expression, suggesting partially impaired MoDC maturation, induced regulatory T cells (Tregs) while failing to induce CD4+ T cell proliferation, compared to MoDCs infected with non-pathogenic leptospires (L. biflexa serovar Patoc and L. meyeri serovar Ranarum, MoDCs-NP). In contrast, non-pathogenic leptospires enhanced MoDC maturation and induced higher T cell proliferation including IFN-γ-producing CD4+ T cells, indicative of a Th1-type response. Furthermore, pathogenic leptospires induced higher MoDC apoptosis through a cysteine aspartic acid-specific protease-3 (caspase-3)-dependent pathway and upregulated expression of the prostaglandin-endoperoxide synthase 2 (PTGS2) gene. Notably, prostaglandin E2 (PGE2), a product of the PTGS2 pathway, was found at higher levels in the sera of patients with acute leptospirosis and in the supernatant of MoDCs-P, possibly contributing to Treg induction, compared to those of healthy donors and MoDCs-NP, respectively. In conclusion, this study reveals a novel immunosuppressive strategy employed by pathogenic Leptospira to evade host immunity by partially impairing MoDC maturation and inducing Tregs. These findings deepen our understanding of leptospirosis pathogenesis in humans and may provide a novel strategy to modulate DCs for the prevention and treatment of the disease.

Proteomic profile of naturally released extracellular vesicles secreted from Leptospira interrogans serovar Pomona in response to temperature and osmotic stresses.

Bacterial extracellular vesicles (EVs) are generally formed by pinching off outer membrane leaflets while simultaneously releasing multiple active molecules into the external environment. In this study, we aimed to identify the protein cargo of leptospiral EVs released from intact leptospires grown under three different conditions: EMJH medium at 30 °C, temperature shifted to 37 °C, and physiologic osmolarity (EMJH medium with 120 mM NaCl). The naturally released EVs observed under transmission electron microscopy were spherical in shape with an approximate diameter of 80-100 nm. Quantitative proteomics and bioinformatic analysis indicated that the EVs were formed primarily from the outer membrane and the cytoplasm. The main functional COG categories of proteins carried in leptospiral EVs might be involved in cell growth, survival and adaptation, and pathogenicity. Relative to their abundance in EVs grown in EMJH medium at 30 °C, 39 and 69 proteins exhibited significant changes in response to the temperature shift and the osmotic change, respectively. During exposure to both stresses, Leptospira secreted several multifunctional proteins via EVs, while preserving certain virulence proteins within whole cells. Therefore, leptospiral EVs may serve as a decoy structure for host responses, whereas some virulence factors necessary for direct interaction with the host environment are reserved in leptospiral cells. This knowledge will be useful for understanding the pathogenesis of leptospirosis and developing as one of vaccine platforms against leptospirosis in the future.

Immunogenicity and protective efficacy of SARS-CoV-2 mRNA vaccine encoding secreted non-stabilized spike in female mice.

Establishment of an mRNA vaccine platform in low- and middle-income countries (LMICs) is important to enhance vaccine accessibility and ensure future pandemic preparedness. Here, we describe the preclinical studies of "ChulaCov19", a SARS-CoV-2 mRNA encoding prefusion-unstabilized ectodomain spike protein encapsulated in lipid nanoparticles (LNP). In female BALB/c mice, ChulaCov19 at 0.2, 1, 10, and 30 µg elicits robust neutralizing antibody (NAb) and T cell responses in a dose-dependent relationship. The geometric mean titers (GMTs) of NAb against wild-type (WT, Wuhan-Hu1) virus are 1,280, 11,762, 54,047, and 62,084, respectively. Higher doses induce better cross-NAb against Delta (B.1.617.2) and Omicron (BA.1 and BA.4/5) variants. This elicited immunogenicity is significantly higher than those induced by homologous CoronaVac or AZD1222 vaccination. In a heterologous prime-boost study, ChulaCov19 booster dose generates a 7-fold increase of NAb against Wuhan-Hu1 WT virus and also significantly increases NAb response against Omicron (BA.1 and BA.4/5) when compared to homologous CoronaVac or AZD1222 vaccination. Challenge studies show that ChulaCov19 protects human-ACE-2-expressing female mice from COVID-19 symptoms, prevents viremia and significantly reduces tissue viral load. Moreover, anamnestic NAb response is undetectable in challenge animals. ChulaCov19 is therefore a promising mRNA vaccine candidate either as a primary or boost vaccination and has entered clinical development.

Identification of a novel gut microbiota signature associated with colorectal cancer in Thai population.

Colorectal cancer (CRC) is the third most common cancer worldwide. Dysbiosis of human gut microbiota has been linked to sporadic CRC. This study aimed to compare the gut microbiota profiles of 80 Thai volunteers over 50 years of age among 25 CRC patients, 33 patients with adenomatous polyp, and 22 healthy controls. The 16S rRNA sequencing was utilized to characterize the gut microbiome in both mucosal tissue and stool samples. The results revealed that the luminal microbiota incompletely represented the intestinal bacteria at the mucus layer. The mucosal microbiota in beta diversity differed significantly among the three groups. The stepwise increase of Bacteroides and Parabacteroides according to the adenomas-carcinomas sequence was found. Moreover, linear discriminant analysis effect size showed a higher level of Erysipelatoclostridium ramosum (ER), an opportunistic pathogen in the immunocompromised host, in both sample types of CRC patients. These findings indicated that the imbalance of intestinal microorganisms might involve in CRC tumorigenesis. Additionally, absolute quantitation of bacterial burden by quantitative real-time PCR (qPCR) confirmed the increasing ER levels in both sample types of cancer cases. Using ER as a stool-based biomarker for CRC detection by qPCR could predict CRC in stool samples with a specificity of 72.7% and a sensitivity of 64.7%. These results suggested ER might be a potential noninvasive marker for CRC screening development. However, a larger sample size is required to validate this candidate biomarker in diagnosing CRC.

Vitamin D (1α,25(OH)2D3) supplementation minimized multinucleated giant cells formation and inflammatory response during Burkholderia pseudomallei infection in human lung epithelial cells.

Melioidosis is an infectious disease with high mortality rates in human, caused by the bacterium Burkholderia pseudomallei. As an intracellular pathogen, B. pseudomallei can escape from the phagosome and induce multinucleated giant cells (MNGCs) formation resulting in antibiotic resistance and immune evasion. A novel strategy to modulate host response against B. pseudomallei pathogenesis is required. In this study, an active metabolite of vitamin D3 (1α,25-dihydroxyvitamin D3 or 1α,25(OH)2D3) was selected to interrupt pathogenesis of B. pseudomallei in a human lung epithelium cell line, A549. The results demonstrated that pretreatment with 10-6 M 1α,25(OH)2D3 could reduce B. pseudomallei internalization to A549 cells at 4 h post infection (P < 0.05). Interestingly, the presence of 1α,25(OH)2D3 gradually reduced MNGC formation at 8, 10 and 12 h compared to that of the untreated cells (P < 0.05). Furthermore, pretreatment with 10-6 M 1α,25(OH)2D3 considerably increased hCAP-18/LL-37 mRNA expression (P < 0.001). Additionally, pro-inflammatory cytokines, including MIF, PAI-1, IL-18, CXCL1, CXCL12 and IL-8, were statistically decreased (P < 0.05) in 10-6 M 1α,25(OH)2D3-pretreated A549 cells by 12 h post-infection. Taken together, this study indicates that pretreatment with 10-6 M 1α,25(OH)2D3 has the potential to reduce the internalization of B. pseudomallei into host cells, decrease MNGC formation and modulate host response during B. pseudomallei infection by minimizing the excessive inflammatory response. Therefore, 1α,25(OH)2D3 supplement may provide an effective supportive treatment for melioidosis patients to combat B. pseudomallei infection and reduce inflammation in these patients.

Sublethal infection of C3H/HeNJ against Leptospira interrogans serovar Pomona.

Leptospirosis is a worldwide zoonotic disease caused by pathogenic Leptospira spp. Leptospires can infect a variety of mammalian species. Golden Syrian hamsters are mostly used to study acute leptospirosis. However, the immunopathogenic mechanism is poorly understood because immunological reagents for hamsters are limited. This study aimed to establish C3H/HeNJ mice as an animal model for leptospirosis. Five-week-old C3H/HeNJ mice were infected with either low (103 cells) or high (106 cells) inoculum dose of Leptospira interrogans serovar Pomona. All mice were investigated for survival rate, leptospiral load and histopathology of target organs, antibody levels, and cytokine production (IFN-γ, IL-6 and IL-10) at day 28 post-infection. All infected mice survived and did not develop acute lethal infection. However, C3H/HeNJ mice infected with 106 cells of leptospires showed kidney colonization of leptospires and pathological changes in the lung and kidney including renal fibrosis. The glomerular size in PAS-D stained kidney tissues of C3H/HeNJ mice infected with 106 cells of leptospires was significantly reduced compared to that of mice infected with 103 cells of leptospires and non-infected mice. High-dose leptospires induced significantly greater levels of IFN-gamma and IL-6 than low-dose leptospires, but IL-10 level was not significantly different. Moreover, 106 leptospiral cells induced predominant IgG2a isotype suggesting Th1-like response. These results suggest that C3H/HeNJ mice may be used as a sublethal model of leptospirosis.

Resistance-Based Lateral Flow Immunosensor with a NFC-Enabled Smartphone for Rapid Diagnosis of Leptospirosis in Clinical Samples.

Leptospirosis is one of the most life-threatening tropical diseases caused by pathogenic Leptospira. To date, a diagnostic device that offers rapid and sensitive detection of leptospires has been still in demand for proper treatment to reduce the mortality rate. Herein, we create a resistance-based lateral flow immunosensor diagnosis device (R-LFI) that integrates near-field communication (NFC) with a portable smartphone for leptospiral detection in clinical samples. A specific monoclonal antibody against the pathogen was coated on a nitrocellulose membrane (NCM) where the test line was collocated. Two electrodes with a sandwich-like configuration were installed employing a conductive double-sided adhesive tape and connected with a NFC smartphone-based detection system. A half-sandwich immunocomplex formation induced high proton conduction, resulting in a considerable decrement in resistive response. The performance of the R-LFI sensor was evaluated using recombinant LipL32 (rLipL32), Leptospira interrogans, and clinical samples. The R-LFI device exhibited linear responses toward rLipL32 protein in phosphate buffer and L. interrogans-spiked healthy human serum samples within the concentration ranging from 1 to 1000 ng mL-1 (limit of detection (LOD): 0.29 ng mL-1) and from 104 to 106 cell mL-1 (LOD: 4.89 × 103 cell mL-1), respectively. Our R-LFI sensor successfully detected L. interrogans-positive clinical samples as confirmed by polymerase chain reaction (PCR). This platform offers high specificity, selectivity, simplicity, miniscule sample volume, and no labeling element requirement. These desirable features make it particularly suitable for countries where medical facilities and resources are limited.

High prevalence of mgrB-mediated colistin resistance among carbapenem-resistant Klebsiella pneumoniae is associated with biofilm formation, and can be overcome by colistin-EDTA combination therapy.

The global prevalence of colistin-resistant Klebsiella pneumoniae (ColRkp) facilitated by chromosomal and plasmid-mediated Ara4N or PEtN-remodeled LPS alterations has steadily increased with increased colistin usage for treating carbapenem-resistant K. pneumoniae (CRkp). Our study demonstrated the rising trend of ColRkp showing extensively and pandrug-resistant characteristics among CRkp, with a prevalence of 28.5%, which was mediated by chromosomal mgrB, pmrB, or phoQ mutations (91.5%), and plasmid-mediated mcr-1.1, mcr-8.1, mcr-8.2 alone or in conjunction with R256G PmrB (8.5%). Several genetic alterations in mgrB (85.1%) with increased expressions of Ara4N-related phoPQ and pmrK were critical for establishing colistin resistance in our isolates. In this study, we discovered the significant associations between extensively drug-resistant bacteria (XDR) and pandrug-resistant bacteria (PDR) ColRkp in terms of moderate, weak or no biofilm-producing abilities, and altered expressions of virulence factors. These ColRkp would therefore be very challenging to treat, emphasizing for innovative therapy to combat these infections. Regardless of the underlying colistin-resistant mechanisms, colistin-EDTA combination therapy in this study produced potent synergistic effects in both in vitro and in vivo murine bacteremia, with no ColRkp regrowth and improved animal survival, implying the significance of colistin-EDTA combination therapy as systemic therapy for unlocking colistin resistance in ColRkp-associated bacteremia.

Designing Adjuvant Formulations to Promote Immunogenicity and Protective Efficacy of Leptospira Immunoglobulin-Like Protein A Subunit Vaccine.

The leptospirosis burden on humans, especially in high-risk occupational groups and livestock, leads to public health and economic problems. Leptospirosis subunit vaccines have been under development and require further improvement to provide complete protection. Adjuvants can be used to enhance the amplitude, quality, and durability of immune responses. Previously, we demonstrated that LMQ adjuvant (neutral liposomes containing monophosphoryl lipid A (MPL) and Quillaja saponaria derived QS21 saponin) promoted protective efficacy of LigAc vaccine against Leptospira challenge. To promote immunogenicity and protective efficacy of the subunit vaccines, three alternative adjuvants based on neutral liposomes or squalene-in-water emulsion were evaluated in this study. LQ and LQuil adjuvants combined the neutral liposomes with the QS21 saponin or Quillaja saponaria derived QuilA® saponin, respectively. SQuil adjuvant combined a squalene-in-water emulsion with the QuilA® saponin. The immunogenicity and protective efficacy of LigAc (20 µg) formulated with the candidate adjuvants were conducted in golden Syrian hamsters. Hamsters were vaccinated three times at a 2-week interval, followed by a homologous challenge of L. interrogans serovar Pomona. The results showed that LigAc combined with LQ, LQuil, or SQuil adjuvants conferred substantial antibody responses and protective efficacy (survival rate, pathological change, and Leptospira renal colonization) comparable to LMQ adjuvant. The LigAc+LQ formulation conferred 62.5% survival but was not significantly different from LigAc+LMQ, LigAc+LQuil, and LigAc+SQuil formulations (50% survival). This study highlights the potential of saponin-containing adjuvants LMQ, LQ, LQuil, and SQuil for both human and animal leptospirosis vaccines.

Surface proteomics and label-free quantification of Leptospira interrogans serovar Pomona.

Leptospirosis is a re-emerging zoonosis with a global distribution. Surface-exposed outer membrane proteins (SE-OMPs) are crucial for bacterial-host interactions. SE-OMPs locate and expose their epitope on cell surface where is easily accessed by host molecules. This study aimed to screen for surface-exposed proteins and their abundance profile of pathogenic Leptospira interrogans serovar Pomona. Two complementary approaches, surface biotinylation and surface proteolytic shaving, followed by liquid chromatography tandem-mass spectrometry (LC-MS/MS) were employed to identify SE-OMPs of intact leptospires. For quantitative comparison, in-depth label-free analysis of SE-OMPs obtained from each method was performed using MaxQuant. The total number of proteins identified was 1,001 and 238 for surface biotinylation and proteinase K shaving, respectively. Among these, 39 were previously known SE-OMPs and 68 were predicted to be localized on the leptospiral surface. Based on MaxQuant analysis for relative quantification, six known SE-OMPs including EF- Tu, LipL21, LipL41, LipL46, Loa22, and OmpL36, and one predicted SE-OMPs, LipL71 were found in the 20 most abundant proteins, in which LipL41 was the highest abundant SE-OMP. Moreover, uncharacterized LIC14011 protein (LIP3228 ortholog in serovar Pomona) was identified as a novel predicted surface ßb-OMP. High-abundance leptospiral SE-OMPs identified in this study may play roles in virulence and infection and are potential targets for development of vaccine or diagnostic tests for leptospirosis.

Novel colistin-EDTA combination for successful eradication of colistin-resistant Klebsiella pneumoniae catheter-related biofilm infections.

Development of an effective therapy to overcome colistin resistance in Klebsiella pneumoniae, a common pathogen causing catheter-related biofilm infections in vascular catheters, has become a serious therapeutic challenge that must be addressed urgently. Although colistin and EDTA have successful roles for eradicating biofilms, no in vitro and in vivo studies have investigated their efficacy in catheter-related biofilm infections of colistin-resistant K. pneumoniae. In this study, colistin resistance was significantly reversed in both planktonic and mature biofilms of colistin-resistant K. pneumoniae by a combination of colistin (0.25-1 µg/ml) with EDTA (12 mg/ml). This novel colistin-EDTA combination was also demonstrated to have potent efficacy in eradicating colistin-resistant K. pneumoniae catheter-related biofilm infections, and eliminating the risk of recurrence in vivo. Furthermore, this study revealed significant therapeutic efficacy of colistin-EDTA combination in reducing bacterial load in internal organs, lowering serum creatinine, and protecting treated mice from mortality. Altered in vivo expression of different virulence genes indicate bacterial adaptive responses to survive in hostile environments under different treatments. According to these data discovered in this study, a novel colistin-EDTA combination provides favorable efficacy and safety for successful eradication of colistin-resistant K. pneumonia catheter-related biofilm infections.

Identification of in vivo expressed proteins in live attenuated lipopolysaccharide mutant that mediates heterologous protection against Leptospira spp.

Leptospirosis vaccines that elicit broad protection against a range of pathogenic Leptospira spp. would overcome a major drawback of currently licensed bacterin vaccines. Live attenuated vaccine produced from a lipopolysaccharide (LPS) mutant strain of L. interrogans serovar Manilae M1352 (Live M1352) stimulated better protective efficacy than heat killed M1352 (HK M1352) against a heterologous challenge with L. interrogans serovar Pomona. To identify antigens of Live M1352 potentially responsible for cross protection, in vivo-induced antigen technology (IVIAT), a powerful tool to identify in vivo-induced (ivi) genes expressed during infection, was employed in this study. Pooled sera from hamsters immunized with Live M1352 were sequentially adsorbed with various preparations of in vitro grown M1352. The pre-adsorbed sera were used to screen a genomic expression library of M1352. Nineteen strongly reactive clones were selected for DNA sequencing. These ivi genes are conserved in most Leptospira strains. Four selected genes including LIMLP_04965 (tolB), LIMLP_01535, LIMLP_06785 (fliI), and LIMLP_14930 were confirmed for their upregulated expression in kidneys of infected hamsters by RT-qPCR, suggesting their role in leptospiral infection. These ivi proteins represent potential targets for vaccine candidates that warrant further investigation for their protective efficacy.

DNA Vaccine Administered by Cationic Lipoplexes or by In Vivo Electroporation Induces Comparable Antibody Responses against SARS-CoV-2 in Mice.

In view of addressing the global necessity of an effective vaccine in the SARS-CoV-2 pandemic, a plasmid DNA vaccine, expressing for the spike (S) protein and formulated in lipoplexes, was manufactured and tested for in vitro transfection and in vivo immunogenicity. Blank cationic liposomes of 130.9 ± 5.8 nm in size and with a zeta potential of +48 ± 12 mV were formulated using the thin-film layer rehydration method. Liposomes were complexed with pCMVkan-S at different N/P ratios. Ratios of 0.25:1 and 1:1 were selected according to their complex stability and controlled size compared to other ratios and tested in vitro for transfection studies and in vivo for immunogenicity. Both selected formulations showed enhanced neutralizing antibody responses compared to pCMVkan-S injected alone, as well as an increased T cell response. The titers observed were similar to those of intramuscular electroporation (IM-EP), which was set as an efficacy goal.

DNA vaccine candidate encoding SARS-CoV-2 spike proteins elicited potent humoral and Th1 cell-mediated immune responses in mice.

More than 65 million people have been confirmed infection with SARS-CoV-2 and more than 1 million have died from COVID-19 and this pandemic remains critical worldwide. Effective vaccines are one of the most important strategies to limit the pandemic. Here, we report a construction strategy of DNA vaccine candidates expressing full length wild type SARS-CoV-2 spike (S) protein, S1 or S2 region and their immunogenicity in mice. All DNA vaccine constructs of pCMVkan-S, -S1 and -S2 induced high levels of specific binding IgG that showed a balance of IgG1/IgG2a response. However, only the sera from mice vaccinated with pCMKkan-S or -S1 DNA vaccines could inhibit viral RBD and ACE2 interaction. The highest neutralizing antibody (NAb) titer was found in pCMVkan-S group, followed by -S1, while -S2 showed the lowest PRNT50 titers. The geometric mean titers (GMTs) were 2,551, 1,005 and 291 for pCMVkan-S, -S1 and -S2, respectively. pCMVkan-S construct vaccine also induced the highest magnitude and breadth of T cells response. Analysis of IFN-γ positive cells after stimulation with SARS-CoV-2 spike peptide pools were 2,991, 1,376 and 1,885 SFC/106 splenocytes for pCMVkan-S, -S1 and -S2, respectively. Our findings highlighted that full-length S antigen is more potent than the truncated spike (S1 or S2) in inducing of neutralizing antibody and robust T cell responses.

A Comparison of Intramuscular and Subcutaneous Administration of LigA Subunit Vaccine Adjuvanted with Neutral Liposomal Formulation Containing Monophosphoryl Lipid A and QS21.

Leptospirosis vaccines with higher potency and reduced adverse effects are needed for human use. The carboxyl terminal domain of leptospiral immunoglobulin like protein A (LigAc) is currently the most promising candidate antigen for leptospirosis subunit vaccine. However, LigAc-based vaccines were unable to confer sterilizing immunity against Leptospira infection in animal models. Several factors including antigen properties, adjuvant, delivery system, and administration route need optimization to maximize vaccine efficacy. Our previous report demonstrated protective effects of the recombinant LigAc (rLigAc) formulated with liposome-based adjuvant, called LMQ (neutral liposome combined with monophosphoryl lipid A and Quillaja saponaria fraction 21) in hamsters. This study aimed to evaluate the impact of two commonly used administration routes, intramuscular (IM) and subcutaneous (SC), on immunogenicity and protective efficacy of rLigAc-LMQ administrated three times at 2-week interval. Two IM vaccinations triggered significantly higher levels of total anti-rLigAc IgG than two SC injections. However, comparable IgG titers and IgG2/IgG1 ratio was observed for both routes after the third immunization. The route of vaccine administration did not influence the survival rate (60%) and renal colonization against lethal Leptospira challenge. Importantly, the kidneys of IM group showed no pathological lesions while the SC group showed mild damage. In conclusion, IM vaccination with rLigAc-LMQ not only elicited faster antibody production but also protected from kidney damage following leptospiral infection better than SC immunization. However, both tested routes did not influence protective efficacy in terms of survival rate and the level of renal colonization.

Reduced Renal Colonization and Enhanced Protection by Leptospiral Factor H Binding Proteins as a Multisubunit Vaccine Against Leptospirosis in Hamsters.

Subunit vaccines conferring complete protection against leptospirosis are not currently available. The interactions of factor H binding proteins (FHBPs) on pathogenic leptospires and host factor H are crucial for immune evasion by inhibition of complement-mediated killing. The inhibition of these interactions may be a potential strategy to clear leptospires in the host. This study aimed to evaluate a multisubunit vaccine composed of four known leptospiral FHBPs: LigA domain 7-13 (LigAc), LenA, LcpA, and Lsa23, for its protective efficacy in hamsters. The mono and multisubunit vaccines formulated with LMQ adjuvant, a combination of neutral liposome, monophosphoryl lipid A, and Quillaja saponaria fraction 21, induced high and comparable specific antibody (IgG) production against individual antigens. Hamsters immunized with the multisubunit vaccine showed 60% survival following the challenge by 20 LD50 of Leptospira interrogans serovar Pomona. No significant difference in survival rate and pathological findings of target organs was observed after vaccinations with multisubunit or mono-LigAc vaccines. However, the multisubunit vaccine significantly reduced leptospiral burden in surviving hamsters in comparison with the monosubunit vaccines. Therefore, the multisubunit vaccine conferred partial protection and reduced renal colonization against virulence Leptospira infection in hamsters. Our multisubunit formulation could represent a promising vaccine against leptospirosis.

A predicted cation transporter protein, BPSS1228, is involved in intracellular behaviour of Burkholderia pseudomallei in a human lung epithelial cell line (A549).

Burkholderia pseudomallei causes melioidosis, a potentially fatal infectious disease in tropical and subtropical countries worldwide. The intracellular behaviour of this pathogen in host cells has been reported to impact the severity of melioidosis, including the development of septicaemia, a consequence of pneumonia melioidosis. We previously identified a predicted cation transporter protein, BPSS1228, that participates in the transitional stage of this intracellular pathogen. For further analysis, in this study B. pseudomallei bpss1228 mutant and complemented strains were constructed and bacterial infectivity on human lung epithelial cells, A549, investigated in vitro Burkholderia pseudomallei bpss1228 mutant showed impaired bacterial adhesion and invasion into A549 cells compared with wild-type strain, while the deficient phenotypes were restored to wild-type levels by the complemented strain. Additionally, the inactivation of bpss1228 in the mutant strain affected flagella-based swimming on a semi-solid surface and resistance to acid stresses simulating intracellular environments. These observations of BPSS1228 relating to B. pseudomallei infection strategies shed a new light on its association with intracellular B. pseudomallei during the interaction with host cells.

Transcription level analysis of intracellular Burkholderia pseudomallei illustrates the role of BPSL1502 during bacterial interaction with human lung epithelial cells.

Melioidosis caused by Burkholderia pseudomallei is a globally important disease of increasing concern according to high case-fatality rate and epidemic spreading. The ability of B. pseudomallei to attach and invade host cells and subsequently survive intracellularly has stimulated many questions concerning the comprehension of bacterial pathogenesis progression. Transcription levels of intracellular B. pseudomallei genes in human lung epithelial cells were therefore analyzed using bioinformatic tools, RT-PCR and real time RT-PCR. Here, it is reported that the identification of bpsl1502, encoding B. pseudomallei SurE (stationary phase survival protein E) located in a global transcriptional regulation operon was accomplished. The up-regulation of B. pseudomallei SurE was demonstrated during intracellular survival of A549 cells at 12, 18, and 24 h post-infection. To investigate the role of this protein, a B. pseudomallei SurE defective mutant was constructed. The invasion and initial survival of the SurE mutants within the A549 cells were impaired. There was no difference, however, between the growth of B. pseudomallei SurE mutant as compared to the wild type in Luria-Bertani culture. These data suggest that SurE may assist B. pseudomallei host cells invade and facilitate early intracellular infection but is not crucial during the stationary growth phase. The identification of B. pseudomallei SurE provides more information of bacterial strategy during an early step of the pathogenesis process of melioidosis.