- Volume 168, Issue 3, 2022

Pore-forming toxins (PFTs) are widely distributed in both Gram-negative and Gram-positive bacteria. PFTs can act as virulence factors that bacteria utilise in dissemination and host colonisation or, alternatively, they can be employed to compete with rival microbes in polymicrobial niches. PFTs transition from a soluble form to become membrane-embedded by undergoing large conformational changes. Once inserted, they perforate the membrane, causing uncontrolled efflux of ions and/or nutrients and dissipating the protonmotive force (PMF). In some instances, target cells intoxicated by PFTs display additional effects as part of the cellular response to pore formation. Significant progress has been made in the mechanistic description of pore formation for the different PFTs families, but in several cases a complete understanding of pore structure remains lacking. PFTs have evolved recognition mechanisms to bind specific receptors that define their host tropism, although this can be remarkably diverse even within the same family. Here we summarise the salient features of PFTs and highlight where additional research is necessary to fully understand the mechanism of pore formation by members of this diverse group of protein toxins.

N -glycolylneuraminic acid (Neu5Gc), and its precursor N-acetylneuraminic acid (Neu5Ac), commonly referred to as sialic acids, are two of the most common glycans found in mammals. Humans carry a mutation in the enzyme that converts Neu5Ac into Neu5Gc, and as such, expression of Neu5Ac can be thought of as a ‘human specific’ trait. Bacteria can utilize sialic acids as a carbon and energy source and have evolved multiple ways to take up sialic acids. In order to generate free sialic acid, many bacteria produce sialidases that cleave sialic acid residues from complex glycan structures. In addition, sialidases allow escape from innate immune mechanisms, and can synergize with other virulence factors such as toxins. Human-adapted pathogens have evolved a preference for Neu5Ac, with many bacterial adhesins, and major classes of toxin, specifically recognizing Neu5Ac containing glycans as receptors. The preference of human-adapted pathogens for Neu5Ac also occurs during biosynthesis of surface structures such as lipo-oligosaccharide (LOS), lipo-polysaccharide (LPS) and polysaccharide capsules, subverting the human host immune system by mimicking the host. This review aims to provide an update on the advances made in understanding the role of sialic acid in bacteria-host interactions made in the last 5–10 years, and put these findings into context by highlighting key historical discoveries. We provide a particular focus on ‘molecular mimicry’ and incorporation of sialic acid onto the bacterial outer-surface, and the role of sialic acid as a receptor for bacterial adhesins and toxins.

It is now 75 years since Marjory Stephenson became the second President of the Society for General Microbiology (SGM). Around the time of her death at the end of 1948 many articles appeared extolling Marjory Stephenson’s contribution to the fields of Biochemistry and Microbiology. Not that much has been written about her since that time, which is unfortunate. Therefore, this brief review is intended as a form of redress and aims to highlight the role of this remarkable scientist in establishing the Society and in promoting Microbiology as a discipline. Notwithstanding the significance of these achievements, however, it is her overall impact on the field of ‘Chemical Microbiology’ and what she achieved through her research that are extraordinary, even by today’s standards. Marjory Stephenson recognized that in order to understand a biological system, the ‘whole’ organism must be considered and this can only be achieved by adopting an interdisciplinary approach: inorganic and organic chemistry, biochemistry, genetics, metabolism and ultimately physiology. Her scientific ethos serves today as a beacon for how scientific research should be conducted, and what we as scientists can learn about how to inspire and mentor the next generation. It is impossible to overstate Marjory Stephenson’s scientific legacy, or her overall contribution to Microbiology.

Bacterial cell envelopes are compositionally complex and crowded and while highly dynamic in some areas, their molecular motion is very limited, to the point of being almost static in others. Therefore, it is no real surprise that studying them at high resolution across a range of temporal and spatial scales requires a number of different techniques. Details at atomistic to molecular scales for up to tens of microseconds are now within range for molecular dynamics simulations. Here we review how such simulations have contributed to our current understanding of the cell envelopes of Gram-negative bacteria.

Burkholderia cenocepacia infections are difficult to treat and there is an urgent need for alternative (combination) treatments. The use of anti-virulence therapies in combination with antibiotics is a possible strategy to increase the antimicrobial susceptibility of the pathogen and to slow down the development of resistance. In the present study we evaluated the β-lactam and colistin-potentiating activity, and anti-virulence effect of the non-mevalonate pathway inhibitor FR900098 against B. cenocepacia in various in vitro and in vivo models. In addition, we evaluated whether repeated exposure to FR900098 alone or when combined with ceftazidime leads to increased resistance. FR900098 potentiated the activity of colistin and several β-lactam antibiotics (aztreonam, cefepime, cefotaxime, ceftazidime, mecillinam and piperacillin) but not of imipenem and meropenem. When used alone or in combination with ceftazidime, FR900098 increased the survival of infected Galleria mellonella and Caenorhabditis elegans. Furthermore, combining ceftazidime with FR900098 resulted in a significant inhibition of the biofilm formation of B. cenocepacia . Repeated exposure to FR900098 in the C. elegans infection model did not lead to decreased activity, and the susceptibility of the evolved B. cenocepacia HI2424 lineages to ceftazidime, FR900098 and the combination of both remained unchanged. In conclusion, FR900098 reduces B. cenocepacia virulence and potentiates ceftazidime in an in vivo C. elegans model, and this activity is not lost during the experimental evolution experiment carried out in the present study.

Escherichia coli is a facultative anaerobe that can grow in a variety of environmental conditions. In the complete absence of O2, E. coli can perform a mixed-acid fermentation that contains within it an elaborate metabolism of formic acid. In this study, we use cavity-enhanced Raman spectroscopy (CERS), FTIR, liquid Raman spectroscopy, isotopic labelling and molecular genetics to make advances in the understanding of bacterial formate and H2 metabolism. It is shown that, under anaerobic (anoxic) conditions, formic acid is generated endogenously, excreted briefly from the cell, and then taken up again to be disproportionated to H2 and CO2 by formate hydrogenlyase (FHL-1). However, exogenously added D-labelled formate behaves quite differently from the endogenous formate and is taken up immediately, independently, and possibly by a different mechanism, by the cell and converted to H2 and CO2. Our data support an anion-proton symport model for formic acid transport. In addition, when E. coli was grown in a micro-aerobic (micro-oxic) environment it was possible to analyse aspects of formate and O2 respiration occurring alongside anaerobic metabolism. While cells growing under micro-aerobic conditions generated endogenous formic acid, no H2 was produced. However, addition of exogenous formate at the outset of cell growth did induce FHL-1 biosynthesis and resulted in formate-dependent H2 production in the presence of O2.

Type IV pili are involved in adhesion, twitching motility, aggregation, biofilm formation and virulence in a variety of Gram-negative bacteria. Burkholderia pseudomallei, the causative agent of melioidosis and a Tier 1 biological select agent, is a Gram-negative bacterium with eight type IV pili-associated loci (TFP1 to TFP8). Most have not been fully characterized. In this study, we investigated BPSS2185, an uncharacterized TFP8 gene that encodes a type IVB pilus protein subunit. Using genetic deletion and complementation analysis in B. pseudomallei JW270, we demonstrate that BPSS2185 plays an important role in twitching motility and adhesion to A549 human alveolar epithelial cells. Compared to JW270, the JW270 ΔBPSS2185 mutant failed to display twitching motility and did not adhere to the epithelial cells. These phenotypes were partially reversed by the complementation of BPSS2185 in the mutant strain. The study also shows that BPSS2185 is expressed only during the onset of mature biofilm formation and at the dispersal of a biofilm, suggesting that the motility characteristic is required to form a biofilm. Our study is the first to suggest that the BPSS2185 gene in TFP8 contributes to twitching motility, adhesion and biofilm formation, indicating that the gene may contribute to B. pseudomallei virulence.

Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) definitive phage type 104 (DT104), S. Worthington, and S. bongori produce ArtAB toxin, which catalyses ADP-ribosylation of pertussis toxin-sensitive G protein. ArtAB gene (artAB) is encoded on a prophage in Salmonella , and prophage induction by SOS-inducing agents is associated with increases in ArtAB production in vitro. However, little is known about the expression of artAB in vivo. Here, we showed a significant increase in artAB transcription of DT104 within macrophage-like RAW264.7 cells. Intracellular expression of ArtAB was also observed by immunofluorescence staining. The induced expression of artAB in DT104 and S. bongori was enhanced by treatment of RAW264.7 cells with phorbol 12-myristate 13-acetate (PMA), which stimulates the production of reactive oxygen species (ROS); however, such induction was not observed in S. Worthington. Upregulation of oxyR, a major regulator of oxidative stress, and cI, a repressor of prophage induction, was observed in S. Worthington within RAW264.7 cells treated with PMA but not in the DT104 strain. Although the expression of oxyR was increased, artAB was upregulated in S. bongori, which lacks the cI gene in the incomplete artAB-encoded prophage. Taken together, oxidative stress plays a role in the production of artAB toxins in macrophages, and high expression levels of oxyR and cI are responsible for the low expression of artAB. Therefore, strain variation in the level of artAB expression within macrophages could be explained by differences in the oxidative stress response of bacteria and might be reflected in its virulence.

Mycobacteriophage D29 infects species belonging to the genus Mycobacterium including the deadly pathogen Mycobacterium tuberculosis. D29 is a lytic phage, although, related to the lysogenic mycobacteriophage L5. This phage is unable to lysogenize in mycobacteria as it lacks the gene encoding the phage repressor. Infection by many mycobacteriophages cause various changes in the host that ultimately leads to inactivation of the latter. One of the host targets often modified in the process is RNA polymerase. During our investigations with phage D29 infected Mycobacterium smegmatis (Msm) we observed that the promoters from both phage, and to a lesser extent those of the host were found to be more active in cells that were exposed to D29, as compared to the unexposed. Further experiments indicate that the RNA polymerase purified from phage infected cells possessed higher affinity for promoters particularly those that were phage derived. Comparison of the purified RNA polymerase preparations from infected and uninfected cells showed that several ancillary transcription factors, Sigma factor F, Sigma factor H, CarD and RbpA are prominently associated with the RNA polymerase from infected cells. Based on our observations we conclude that the higher activity of RNA polymerase observed in D29 infected cells is due to its increased association with ancillary transcription factors.

The fungal pathogen Aspergillus fumigatus is frequently cultured from the sputum of cystic fibrosis (CF) patients along with the bacterium Pseudomonas aeruginosa. A. fumigatus secretes a range of secondary metabolites, and one of these, gliotoxin, has inhibitory effects on the host immune response. The effect of   P. aeruginosa   culture filtrate (CuF) on fungal growth and gliotoxin production was investigated. Exposure of A. fumigatus hyphae to   P. aeruginosa   cells induced increased production of gliotoxin and a decrease in fungal growth. In contrast, exposure of A. fumigatus hyphae to   P. aeruginosa   CuF led to increased growth and decreased gliotoxin production. Quantitative proteomic analysis was used to characterize the proteomic response of A. fumigatus upon exposure to   P. aeruginosa   CuF. Changes in the profile of proteins involved in secondary metabolite biosynthesis (e.g. gliotoxin, fumagillin, pseurotin A), and changes to the abundance of proteins involved in oxidative stress (e.g. formate dehydrogenase) and detoxification (e.g. thioredoxin reductase) were observed, indicating that the bacterial secretome had a profound effect on the fungal proteome. Alterations in the abundance of proteins involved in detoxification and oxidative stress highlight the ability of A. fumigatus to differentially regulate protein synthesis in response to environmental stresses imposed by competitors such as   P. aeruginosa  . Such responses may ultimately have serious detrimental effects on the host.