The innate interferon gamma response of BALB/c and C57BL/6 mice to in vitro Burkholderia pseudomallei infection

BACKGROUND: Burkholderia pseudomallei is the causative agent for melioidosis. For many bacterial infections, cytokine dysregulation is one of the contributing factors to the severe clinical outcomes in the susceptible hosts. The C57BL/6 and BALB/c mice have been established as a differential model of susceptibility in murine melioidosis. In this study, we compared the innate IFN-γ response to B. pseudomallei between the C57BL/6 and BALB/c splenocytes and characterized the hyperproduction of IFN-γ in the relatively susceptible BALB/c mice in vitro. RESULTS: Naïve BALB/c splenocytes were found to produce more IFN-γ in response to live bacterial infection compared to C57BL/6 splenocytes. Natural killer cells were found to be the major producers of IFN-γ, while T cells and Gr-1(intermediate )cells also contributed to the IFN-γ response. Although anti-Gr-1 depletion substantially reduced the IFN-γ response, this was not due to the contribution of Gr-1(high), Ly-6G expressing neutrophils. We found no differences in the cell types making IFN-γ between BALB/c and C57BL/6 splenocytes. Although IL-12 is essential for the IFN-γ response, BALB/c and C57BL/6 splenocytes made similar amounts of IL-12 after infection. However, BALB/c splenocytes produced higher proinflammatory cytokines such as IL-1β, TNF-α, IL-6, IL-18 than C57BL/6 splenocytes after infection with B. pseudomallei. CONCLUSION: Higher percentages of Gr-1 expressing NK and T cells, poorer ability in controlling bacteria growth, and higher IL-18 could be the factors contributing to IFN-γ hyperproduction in BALB/c mice

Identification of tomato plant as a novel host model for Burkholderia pseudomallei

<p>Abstract</p> <p>Background</p> <p><it>Burkholderia pseudomallei </it>is the causative agent for melioidosis, a disease with significant mortality and morbidity in endemic regions. Its versatility as a pathogen is reflected in its relatively huge 7.24 Mb genome and the presence of many virulence factors including three Type Three Secretion Systems known as T3SS1, T3SS2 and T3SS3. Besides being a human pathogen, it is able to infect and cause disease in many different animals and alternative hosts such as <it>C. elegans</it>.</p> <p>Results</p> <p>Its host range is further extended to include plants as we demonstrated the ability of <it>B. pseudomallei </it>and the closely related species <it>B. thailandensis </it>to infect susceptible tomato but not rice plants. Bacteria were found to multiply intercellularly and were found in the xylem vessels of the vascular bundle. Disease is substantially attenuated upon infection with bacterial mutants deficient in T3SS1 or T3SS2 and slightly attenuated upon infection with the T3SS3 mutant. This shows the importance of both T3SS1 and T3SS2 in bacterial pathogenesis in susceptible plants.</p> <p>Conclusions</p> <p>The potential of <it>B. pseudomallei </it>as a plant pathogen raises new possibilities of exploiting plant as an alternative host for novel anti-infectives or virulence factor discovery. It also raises issues of biosecurity due to its classification as a potential bioterrorism agent.</p

Type three secretion system-mediated escape of Burkholderia pseudomallei into the host cytosol is critical for the activation of NFκB.

BackgroundBurkholderia pseudomallei is the causative agent of melioidosis, a potentially fatal disease endemic in Southeast Asia and Northern Australia. This Gram-negative pathogen possesses numerous virulence factors including three "injection type" type three secretion systems (T3SSs). B. pseudomallei has been shown to activate NFκB in HEK293T cells in a Toll-like receptor and MyD88 independent manner that requires T3SS gene cluster 3 (T3SS3 or T3SSBsa). However, the mechanism of how T3SS3 contributes to NFκB activation is unknown.ResultsKnown T3SS3 effectors are not responsible for NFκB activation. Furthermore, T3SS3-null mutants are able to activate NFκB almost to the same extent as wildtype bacteria at late time points of infection, corresponding to delayed escape into the cytosol. NFκB activation also occurs when bacteria are delivered directly into the cytosol by photothermal nanoblade injection.ConclusionsT3SS3 does not directly activate NFκB but facilitates bacterial escape into the cytosol where the host is able to sense the presence of the pathogen through cytosolic sensors leading to NFκB activation

Skin models for cutaneous melioidosis reveal Burkholderia infection dynamics at wound's edge with inflammasome activation, keratinocyte extrusion and epidermal detachment.

Funder: Research Centre of Excellence, Mechanobiology InstituteFunder: National Research FoundationFunder: Cambridge-NUS Global Alliance FundABSTRACTMelioidosis is a serious infectious disease endemic in Southeast Asia, Northern Australia and has been increasingly reported in other tropical and subtropical regions in the world. Percutaneous inoculation through cuts and wounds on the skin is one of the major modes of natural transmission. Despite cuts in skin being a major route of entry, very little is known about how the causative bacterium Burkholderia pseudomallei initiates an infection at the skin and the disease manifestation at the skin known as cutaneous melioidosis. One key issue is the lack of suitable and relevant infection models. Employing an in vitro 2D keratinocyte cell culture, a 3D skin equivalent fibroblast-keratinocyte co-culture and ex vivo organ culture from human skin, we developed infection models utilizing surrogate model organism Burkholderia thailandensis to investigate Burkholderia-skin interactions. Collectively, these models show that the bacterial infection was largely limited at the wound's edge. Infection impedes wound closure, triggers inflammasome activation and cellular extrusion in the keratinocytes as a potential way to control bacterial infectious load at the skin. However, extensive infection over time could result in the epidermal layer being sloughed off, potentially contributing to formation of skin lesions

Differential host susceptibility and bacterial virulence factors driving Klebsiella liver abscess in an ethnically diverse population.

Hypervirulent Klebsiella pneumoniae is an emerging cause of community-acquired pyogenic liver abscess. First described in Asia, it is now increasingly recognized in Western countries, commonly afflicting those with Asian descent. This raises the question of genetic predisposition versus geospecific strain acquisition. We leveraged on the Antibiotics for Klebsiella Liver Abscess Syndrome Study (A-KLASS) clinical trial ongoing in ethnically diverse Singapore, to prospectively examine the profiles of 70 patients together with their isolates' genotypic and phenotypic characteristics. The majority of isolates belonged to capsule type K1, a genetically homogenous group corresponding to sequence-type 23. The remaining K2, K5, K16, K28, K57 and K63 isolates as well as two novel cps isolates were genetically heterogeneous. K1 isolates carried higher frequencies of virulence-associated genes including rmpA (regulator of mucoid phenotype A), kfu (Klebsiella ferric uptake transporter), iuc (aerobactin), iro (salmochelin) and irp (yersiniabactin) than non-K1 isolates. The Chinese in our patient cohort, mostly non-diabetic, had higher prevalence of K1 infection than the predominantly diabetic non-Chinese (Malays, Indian and Caucasian). This differential susceptibility to different capsule types among the various ethnic groups suggests patterns of transmission (e.g. environmental source, familial transmission) and/or genetic predisposition unique to each race despite being in the same geographical location

Glutathione ratio affects intracellular bacterial control via the IL-12–IFNγ axis [20].

<p>A decreased glutathione ratio in monocytes results in reduced IL-12 production. IL-12 induces the production of IFNγ from natural killer (NK) and T cells. IFNγ is necessary to activate the infected monocytes to become bactericidal for killing of intracellular <i>B. pseudomallei</i> (<i>B. p.</i>) and <i>M. tuberculosis (M. tb.)</i>. The IL-12 response from monocytes infected with <i>B. p.</i> is dependent on TRIF and MyD88 adaptors, yet these signaling pathways do not seem to be affected by the GSH ratio <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003794#ppat.1003794-Tan1" target="_blank">[20]</a>. Perhaps another signaling pathway mediated by an intracellular pattern recognition receptor (PRR) is also necessary for IL-12 induction, and this pathway is modulated by GSH. The effect of GSH on diabetic NK cell and neutrophil function in response to <i>B. p.</i> is currently under investigation. In tuberculosis, indirect evidence where GSH was added to healthy NK cells and neutrophils in vitro shows increased cellular control of <i>M. tb. </i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003794#ppat.1003794-Morris1" target="_blank">[33]</a>.</p

Modulation of bacterial virulence and fitness by host glutathione

10.1016/j.mib.2018.10.004CURRENT OPINION IN MICROBIOLOGY478-1

New roles for glutathione: Modulators of bacterial virulence and pathogenesis

Low molecular weight (LMW) thiols contain reducing sulfhydryl groups that are important for maintaining antioxidant defense in the cell. Aside from the traditional roles of LMW thiols as redox regulators in bacteria, glutathione (GSH) has been reported to affect virulence and bacterial pathogenesis. The role of GSH in virulence is diverse, including the activation of virulence gene expression and contributing to optimal biofilm formation. GSH can also be converted to hydrogen sulfide (H2S) which is important for the pathogenesis of certain bacteria. Besides GSH, some bacteria produce other LMW thiols such as mycothiol and bacillithiol that affect bacterial virulence. We discuss these newer reported functions of LMW thiols modulating bacterial pathogenesis either directly or indirectly and via modulation of the host immune system

Characterisation of cellular effects of Burkholderia pseudomallei cycle inhibiting factor (Cif)

Cycle inhibiting factors (Cifs) are type III secretion system effectors produced by some Gram-negative pathogenic bacteria including Burkholderia pseudomallei. Through their deamidase activity, Cifs inhibit the activity of Cullin RING E3 ubiquitin ligases (CRL). CRL inhibition induces the accumulation of cell cycle inhibitors p21 and p27, thereby leading to host cell cycle arrest. However, whether Cif exerts additional effects on host cells that are important in bacterial pathogenesis is currently poorly understood. In this study, we found that Cif exerts a bimodal effect on NF-κB signalling. Cif increases basal NF-κB activity. This effect is dependent on Cif-mediated activation of ERK MAPK. On the other hand, Cif inhibits NF-κB activation by TNFα and Burkholderia thailandensis infection. This inhibitory effect on NF-κB activity is partially mediated by Cif-dependent inhibition of CRLs. We also found that Cif only has a modest effect in stimulating the intracellular replication of the B. pseudomallei surrogate, B. thailandensis. The observed Cif-dependent stimulation of B. thailandensis intracellular replication was not, or was only partially, due to CRL inhibition. Furthermore, the increased B. thailandensis replication induced by Cif was independent of ERK MAPK activation. Our findings suggest that Cif likely exerts additional cellular effects through novel targets