Outer membrane protein A (OmpA) deficient Salmonella Typhimurium displays enhanced susceptibility towards β-lactam antibiotics: third-generation cephalosporins (ceftazidime) and carbapenems (meropenem)

The invasive non-typhoidal serovar of Salmonella enterica, namely Salmonella Typhimurium ST313, causes bloodstream infection in sub-Saharan Africa. Like other bacterial pathogens, the development of antimicrobial resistance is a severe problem in curing non-typhoidal Salmonella infection. In this work, we have investigated the role of four prominent outer membrane porins of S. Typhimurium, namely OmpA, OmpC, OmpD, and OmpF, in resistance against broad-spectrum β-lactam antibiotics- ceftazidime and meropenem. We found that deleting OmpA from Salmonella makes the bacteria susceptible to β-lactam drugs. The MIC for both the antibiotics reduced significantly for STM ΔompA compared to the wild-type and the ompA complemented strains. Despite the presence of antibiotics, the uninterrupted growth of STM ΔompC, ΔompD, and ΔompF endorsed the dispensability of these three porins in antimicrobial resistance. The β-lactam antibiotics caused massive depolarization in the outer membrane of the bacteria in the absence of OmpA. We have proved that none of the extracellular loops but the complete structure of perfectly folded OmpA is required by the bacteria for developing antimicrobial resistance. Our data revealed that STM ΔompA consumed more antibiotics than the wild-type and the complemented strain, resulting in severe damage of the bacterial outer membrane and subsequent killing of the pathogen by antibiotic-mediated oxidative stress. Upon deleting ompA, the steady decrease in the relative proportion of antibiotic-resistant persisters and the clearance of the STM ΔompA from the liver and spleen of C57BL/6 mice upon treatment with ceftazidime proved the role of OmpA in rendering protection against β-lactam antibiotics.

Immune Responses and Protective Immunity in Pangasius Pangasius (Hamilton, 1822) as Induced by Outer Membrane Proteins of Edwardsiella Tarda and Aluminium Hydroxide Adjuvant Complex

Edwardsiella tarda is considered one of the important bacterial fish pathogens. The outer membrane proteins (OMPs) of E. tarda are structurally and functionally conserved, and immunogenic. This study assessed the effects of the OMPs of E. tarda CGH9 as a vaccine without aluminium hydroxide [AH] (T1) and with AH adjuvant (T2) on the respiratory burst (ROB) activity, lymphocyte proliferation of head kidney (HK) leukocytes, and serum antibody production in pangas catfish Pangasius pangasius. The ROB activity and lymphocyte proliferation of HK leukocytes increased in both vaccinated groups compared to control. Nonetheless, the T2 group showed a gradual increase in ROB activity and lymphocyte proliferation of HK leukocytes up to 3-weeks post-vaccination (wpv). The serum antibody production in the T1 group decreased initially for up to 2-wpv and increased from 3-wpv; whereas, in the T2 group, the serum-specific antibody levels were significantly high from 1-wpv compared to control. Simultaneously, the protective efficacy in terms of relative percentage survival (RPS) in the T2 group after injecting with a lethal dose of E. tarda CGH9 was high (89.00±15.56) compared to the T1 group (78.00±0.00). Furthermore, the catfish administered with a booster dose of E. tarda OMPs with or without AH adjuvant showed no additional increase in immune response or protective immunity. These results suggested that E. tarda OMPs and AH adjuvant complex has a higher potential to induce protective immunity, which may be a good choice as a vaccine to combat E. tarda infection in catfish.

Dissecting the interactions of PINK1 with the TOM complex in depolarized mitochondria

Mitochondria dysfunction is involved in the pathomechanism of many illnesses including Parkinson's disease. PINK1, which is mutated in some cases of familiar Parkinsonism, is a key component in the degradation of damaged mitochondria by mitophagy. The accumulation of PINK1 on the mitochondrial outer membrane (MOM) of compromised organelles is crucial for the induction of mitophagy, but the molecular mechanism of this process is still unresolved. Here, we investigate the association of PINK1 with the TOM complex. We demonstrate that PINK1 heavily relies on the import receptor TOM70 for its association with mitochondria and directly interacts with this receptor. The structural protein TOM7 appears to play only a moderate role in PINK1 association with the TOM complex, probably due to its role in stabilizing this complex. PINK1 requires the TOM40 pore lumen for its stable interaction with the TOM complex and apparently remains there during its further association with the MOM. Overall, this study provides new insights on the role of the individual TOM subunits in the association of PINK1 with the MOM of depolarized mitochondria.

A bacterial secretosome for regulated envelope biogenesis and quality control

As the first line of defence against antibiotics, the Gram-negative bacterial envelope and its biogenesis are of considerable interest to the microbiological and biomedical communities. All bacterial proteins are synthesised in the cytosol, so inner- and outer-membrane proteins, and periplasmic residents have to be transported to their final destinations via specialised protein machinery. The Sec translocon, a ubiquitous integral inner-membrane (IM) complex, is key to this process as the major gateway for protein transit from the cytosol to the cell envelope; this can be achieved during their translation, or afterwards. Proteins need to be directed to the inner-membrane (usually co-translational), otherwise SecA utilises ATP and the proton-motive-force (PMF) to drive proteins across the membrane post-translationally. These proteins are then picked up by chaperones for folding in the periplasm or delivered to the β-barrel assembly machinery (BAM) for incorporation into the outer-membrane. The core heterotrimeric SecYEG-complex forms the hub for an extensive network of interactions that regulate protein delivery and quality control. Here, we conduct a biochemical exploration of this secretosome: a very large, versatile and inter-changeable assembly with the Sec-translocon at its core; featuring interactions that facilitate secretion (SecDF), inner- and outer-membrane protein insertion (respectively, YidC and BAM), protein folding and quality control (e.g. PpiD, YfgM and FtsH). We propose the dynamic interplay amongst these and other factors act to ensure efficient whole envelope biogenesis, regulated to accommodate the requirements of cell elongation and division. This organisation would be essential for cell wall biogenesis and remodelling and thus its perturbation would be a good strategy for the development of anti-microbials.

An Investigation into the Effects of Outer Membrane Vesicles and Lipopolysaccharide of Porphyromonas gingivalis on Blood-Brain Barrier Integrity, Permeability, and Disruption of Scaffolding Proteins in a Human in vitro Model

Background: The effects of the key pathogens and virulence factors associated with gum disease such as Porphyromonas gingivalis (P. gingivalis) on the central nervous system is of great interest with respect to development of neuropathologies and hence therapeutics and preventative strategies. Chronic infections and associated inflammation are known to weaken the first line of defense for the brain, the blood-brain barrier (BBB). Objective: The focus of this study is to utilize an established human in vitro BBB model to evaluate the effects of P. gingivalis virulence factors lipopolysaccharide (LPS) and outer membrane vesicles (OMVs) on a primary-derived human model representing the neurovascular unit of the BBB. Methods: Changes to the integrity of the BBB after application of P. gingivalis LPS and OMVs were investigated and correlated with transport of LPS. Additionally, the effect of P. gingivalis LPS and OMVs on human brain microvascular endothelial cells in monolayer was evaluated using immunofluorescence microscopy. Results: The integrity of the BBB model was weakened by application of P. gingivalis LPS and OMVs, as measured by a decrease in electrical resistance and a recovery deficit was seen in comparison to the controls. Application of P. gingivalis OMVs to a monoculture of human brain microvascular endothelial cells showed disruption of the tight junction zona occludens protein (ZO-1) compared to controls. Conclusion: These findings show that the integrity of tight junctions of the human BBB could be weakened by association with P. gingivalis virulence factors LPS and OMVs containing proteolytic enzymes (gingipains).

Outer Membrane Vesicles (OMVs) of Pseudomonas aeruginosa Provide Passive Resistance but Not Sensitization to LPS-Specific Phages

Outer membrane vesicles (OMVs) released from gram-negative bacteria are key elements in bacterial physiology, pathogenesis, and defence. In this study, we investigated the role of Pseudomonas aeruginosa OMVs in the anti-phage defence as well as in the potential sensitization to LPS-specific phages. Using transmission electron microscopy, virion infectivity, and neutralization assays, we have shown that both phages efficiently absorb on free vesicles and are unable to infect P. aeruginosa host. Nevertheless, the accompanying decrease in PFU titre (neutralization) was only observed for myovirus KT28 but not podovirus LUZ7. Next, we verified whether OMVs derived from wild-type PAO1 strain can sensitize the LPS-deficient mutant (Δwbpl PAO1) resistant to tested phages. The flow cytometry experiments proved a quite effective and comparable association of OMVs to Δwbpl PAO1 and wild-type PAO1; however, the growth kinetic curves and one-step growth assay revealed no sensitization event of the OMV-associated phage-resistant P. aeruginosa deletant to LPS-specific phages. Our findings for the first time identify naturally formed OMVs as important players in passive resistance (protection) of P. aeruginosa population to phages, but we disproved the hypothesis of transferring phage receptors to make resistant strains susceptible to LPS-dependent phages.