- Volume 4, Issue 5, 2022

The human disease cholera, marked by acute, voluminous watery diarrhea, is caused by the gram-negative, aquatic bacterium Vibrio cholerae. All seven cholera pandemics since 1817 were identified as being caused by just 2 of over 155 known V. choleraeserogroups: O1 and O139. The O1 serogroup is divided into two biotypes: classical and El Tor. Classical biotype is associated with pandemics 1 through 6, but the El Tor biotype has since displaced classical as the causative agent of the ongoing 7th cholera pandemic over the past 60 years. The El Tor genome resembles that of classical but has acquired two unique pathogenicity islands known as Vibrio Seventh Pandemic (VSP) -1 and -2. El Tor biotype has been associated with prolonged colonization, infection, and disease both in humans and in the zebrafish natural host model. The zebrafish model allows for complete observation of Vibrio cholerae infection in a system undisrupted by antibiotic use or immune suppression. El Tor strains colonize the zebrafish intestine for up to 10 days longer than classical strains. Preliminary studies demonstrate VSP-2 is required to observe this phenotype, but VSP-1 is not. By creating targeted regional knockouts of the VSP-2 island, the specific gene(s) essential for enabling prolonged colonization will be identified and applied to the understanding of how El Tor interacts with a natural host. By identifying the genes used by El Tor to colonize a natural host for prolonged periods, we gain insight into how this pathogen may persist in the environment and perpetuate disease.

Neisseria gonorrhoeae (the gonococcus) is the causative agent of the sexually-transmitted infection gonorrhoea, and has developed resistance to all classes of antimicrobials. In gonococci, plasmids can mediate high-level antimicrobial resistance (AMR) to tetracyclines and ß-lactams. Plasmids can spread through bacterial populations by transformation and conjugation, resulting in the rapid dissemination of traits. Characterisation of plasmids, including understanding their distribution in bacterial populations, is therefore key to understanding bacterial evolution, and in particular the spread of AMR. N. gonorrhoeae can harbour three plasmids, conjugative (pConj), ß-lactamase (pbla) and cryptic (pCryp). Using genomic and phylogenetic analyses, we show that plasmids are widespread in a large collection of gonococcal isolates from 56 countries. We found that variants of pConj (which can mediate tetracycline resistance) and pbla expressing TEM-135 ß-lactamase are associated with distinct gonococcal lineages. Furthermore, AMR plasmids are significantly more prevalent in gonococci from less wealthy countries. Over 94% of gonococci possess the cryptic plasmid (pCryp), and its absence can be correlated with the presence of a novel chromosomal Type IV secretion system. Our results reveal the extent of plasmid-mediated AMR in the gonococcus, particularly in less wealthy countries, where diagnostic and therapeutic options can be limited, and highlight the risk of their global spread.

Daptomycin is a last-resort antibiotic for the treatment of invasive diseases caused by methicillin-resistant Staphylococcus aureus. However, despite potent activity in vitro,daptomycin fails to resolve up to 30% of cases of staphylococcal bacteraemia, suggesting that the host environment reduces bacterial susceptibility to the antibiotic. Using human serum as a model of bacteraemia, we demonstrated that the host environment induced daptomycin tolerance via activation of the GraRS two-component system. Testing of various antimicrobial peptides present in serum revealed that the human cathelicidin LL-37 was able to bind GraS, induce GraRS signalling and confer daptomycin tolerance. Activation of GraRS by serum led to daptomycin tolerance via changes in the staphylococcal cell envelope. For example, GraRS-dependent increases in positive surface charge and peptidoglycan content occurred in serum, leading to reduced daptomycin binding. Additionally, incubation in serum led to a Cls2-dependent increase in the cardiolipin content of the membrane which contributed to daptomycin tolerance. Finally, inhibition of both peptidoglycan and cardiolipin synthesis together completely abolished acquisition of daptomycin tolerance in serum, demonstrating that these processes fully explain the tolerance phenotype. In summary, host LL-37 activates staphylococcal GraRS signalling, causing changes in the cell surface which confer daptomycin tolerance. This demonstrates how host defences can compromise antibiotic efficacy, and also provides a rationale for combination therapies to prevent the development of daptomycin tolerance and reduce rates of treatment failure.

Human noroviruses (HNV) are a prevalent cause of gastroenteritis that contribute to >200,000 deaths each year and cost >£40 billion worldwide per annum. There is currently no approved vaccine or therapy, and a greater understanding of the virus life-cycle could help develop new approaches towards disease control. Although HNV infection is usually self-limiting, persistent infections can establish in immunocompromised people - however the underlying mechanisms are poorly understood. Our studies use the murine norovirus (MNV) model system to investigate fundamental virus biology, and several strains of MNV can also persist in the murine host. The primary receptor for MNV, CD300lf, interacts with a network of amino acids (AAs) on the protruding domain of the virus major capsid protein (VP1). We hypothesised that genetic variations leading to changes within this network of AAs could influence the VP1-CD300lf interaction and viral persistence.

Bioinformatic analysis of the VP1-receptor interface highlighted variation in just a single AA that correlates with persistent MNV strains. To confirm this AA is important for receptor interactions we conducted in vitro evolution experiments on suspension or adherent grown cells. Passage through suspension cells resulted in the selection of hydrophobic residues at this position co-incidental with a 1.5-fold increase in viral titre. In contrast, small polar residues were maintained at this position during passage on adherent cells. Furthermore, infectivity assays with infectious clones suggest that hydrophobic residues favour infection of suspension cells over adherent cells. Work is ongoing to understand the importance of this AA on viral infectivity and persistence.

Mycobacterium tuberculosis remains the leading cause of mortality from a single infectious organism, infecting nearly one-third of the global population. The current emergence of multidrug-resistant strains represents a serious health problem nowadays. Moreover, regulation of gene expression through RNA metabolism is a key mechanism for bacterial growth, division and rapid accommodation to environmental conditions. Ribonucleases are enzymes present in all living organisms that play an important role in RNA processing and degradation. Particularly, ribonucleases belonging to the RNB-family are often essential for viability of prokaryotes and are implicated in the establishment of virulence of several pathogens. In the present study we aim to structurally and functionally characterize two putative exoribonucleases from the RNB-family of enzymes inM. tuberculosis. Overexpression and purification of the proteins was performed and further in vitro activity, binding and helicase assays using synthetic RNA substrates were accomplished. In parallel, a biophysical characterization proceeded with several crystallization trials and protein stability tests. We have demonstrated that both RNases are 3’-5’ exoribonucleases with different degradation properties and unravelled the importance of highly conserved residues for catalysis. Moreover, we were able to identify improved buffer formulations that increase protein stability, possibly enhancing their propensity to crystallize. The information regarding RNA metabolism in M. tuberculosis is limited and RNB-family enzymes have not been previously characterized in this important human pathogen. Thus, a complete knowledge of these ribonucleases is an approach to recognize their influence in M. tuberculosis metabolism and to better understand the post-transcriptional control in this pathogen.

The Aedes aegypti mosquito is a major vector of chikungunya virus (CHIKV), which has no licensed vaccine. Engineered mosquitoes expressing long RNA hairpins (lhRNA) or small RNAs against selected arboviruses have been developed to limit virus replication, but their silencing efficiency has not been compared.

We developed lhRNA and short hairpin RNA (shRNA) arrays against CHIKV non-structural protein nsP2. We used a Tet-response element (TRE) and a tTA transactivator to control expression of lhRNAs (TRE-lhRNA) and shRNAs (TRE-shRNA). Constitutive expression in Aag2 cells was assessed with a PUb-tTA driverand midgut specific expression in transgenic mosquitoes was assessed using Carboxypeptidase A (AeCPA)-tTA as driver. In vitrointerference ability was determined with a CHIKV split replication system, and a synthetic luciferase reporter with mRNA containing the targeted CHIKV sequence (CHIK-FF). In vivo interference was tested by inserting the TRE-lhRNA and TRE-shRNA constructs into a AeCPA-tTA line so both constructs expressed from the same locus, and a TRE reporter line that expressed an AmCyan reporter with an N terminal CHIKV fusion (CHIK-AmC).

In Aag2 cells, shRNAs were more effective than lhRNA in silencing both the CHIKV split replication system (99.7%, S. D. ± 0.47 and 72.8%, S. D. ± 9.50, respectively, P<0.001) and CHIK-FF (98.8%, S. D. ± 0.71 and 50.9%, S. D. ± 7.92, respectively, P<0.05). Similar results were observed in transgenic mosquitoes when comparing AmC expression in the midgut.

This study demonstrates that, in mosquitoes, effectively chosen shRNAs can induce greater interference of the desired viral target than the corresponding lhRNA.

Multidrug-resistant pathogens have become a global threat. In this context, filamentous Actinobacteria has been proven to be an exceptional source of antimicrobial metabolites. In particular, rare actinomycetes isolated from marine environments have been proposed as a potential source of yet untapped specialised metabolites. In this study, two novel species, Pseudonocardia abyssalis sp. nov. and Pseudonocardia oceani sp. nov, isolated from deep Southern Ocean sediments are described, both in terms of their phenotypic and genomic characterization. Furthermore, the genomic architecture, with a focus on Biosynthetic Gene Cluster (BGC), across eight strains belonging to the two novel species were investigated. A total of 14 Gene Cluster Families (GCF) were identified, of which five GCFs comprise BGCs from both species, and nine were specific to each species. Moreover, a correlation of GCFs to phylogeny was observed. Following genome analysis, a comparative mass-spectrometry based metabolomics analysis was carried out with one strain from each new species, as well as Pseudonocardia ammonioxydans and Pseudonocardia sediminis, also of marine origin. The metabolomics profiles agreed with the GCF distribution, where a group of ubiquitous metabolites were produced by both new Pseudonocardia spp., while groups of species-specific metabolites were also detected. This metabolic-repertoire was found to be elicitated through the addition of N-acetyl glucosamine (GlcNAc), revealing chemically-inducible bioactivity against the fungi Candida albicas and multidrug-resistant Candida auris. These results showcase the power of a combined genomic-metabolomics approach to investigate rare-actinomycetes from understudied locations and have uncovered a wealth of both biosynthetic and chemical diversity for further investigation.

Many bacterial species secrete polymers and form mat-like structures. These mats can be useful (e.g., in bioremediation), or problematic (e.g., in hospital settings). The molecular bases of mat formation have been investigated in a number of species, including various pseudomonads. One well-characterized example is the plant symbiont Pseudomonas fluorescens SBW25. Laboratory populations of SBW25 readily acquire mutations in one of three regulatory loci (wsp, aws, mws), leading to the over-production of the secondary messenger cyclic-di-GMP. In turn, this activates the production of a cellulose-like polymer, the major structural component of the SBW25 mat. Here, we dissect and compare the molecular mechanisms of mat formation in two further plant-associated pseudomonads: P. simiae PICF7 and P. fluorescens A506. We find that both PICF7 and A506 are capable of mat formation in the laboratory, by distinct molecular routes. Mat formation in PICF7 involves mutations in wsp, aws, or mws that serve to activate the production of Pel (as opposed to cellulose in SBW25). Contrastingly, A506 mat formation does not require mutation of wsp, aws, or mws (despite their retention in the genome). Instead, our results are consistent with a readily reversible, non-mutational route to polymer production and mat formation in A506. Overall, our results demonstrate the presence of multiple molecular routes to mat formation among environmental, plant-associated pseudomonads.

The presented work has since been published: Mukherjee A, Dechow-Seligmann G, Gallie J. 2022. Molecular Microbiology 117(2): 394-410.

Hepatitis E Virus (HEV) is one of the leading causes of acute viral hepatitis, with ~20 million HEV infections worldwide per annum, and mortality rates up to 25% in pregnant women. However, many aspects of the biology of the virus are poorly understood. HEV has a positive-sense single-stranded RNA genome. ORF1 encodes the non-structural polyprotein required for viral RNA replication. This polyprotein (sometimes termed pORF1) is predicted to contain seven domains based on sequence homology to related viruses and it is hypothesised that this polyprotein must undergo proteolysis to generate functional protein units. However, it is unknown if the pORF1 polyprotein undergoes full proteolysis, the potential locations of any cleavage boundaries and whether a viral or host cell protease is responsible.

We have adapted our in vitro-based proteolysis assays to investigate cleavage of HEV pORF1. In comparison to related RNA viruses, our data suggest that pORF1 has no auto-catalytic activity. Previous studies have shown that the liver-produced protease, thrombin, is essential for replication. In the presence of thrombin, we have shown that the ORF1 polyprotein undergoes specific proteolysis to produce eight distinct protein products. Combining bioinformatics with pORF1 truncations/mutagenesis, we have located the position of the pORF1 thrombin cleavage sites. Interestingly, these cleavage sites correspond to the junctions between the predicted pORF1 protein domains. Our data suggests that thrombin is an important cellular protease for controlling pORF1 proteolysis. Work is ongoing to understand the importance of each thrombin cleavage site in viral replication.

Escherichia coli is a Gram-negative bacterium that colonizes the human intestine and virulent strains can cause severe diarrhoeal and extraintestinal diseases. The protein SslE is secreted by a range of pathogenic and some commensal E. colistrains. It can degrade mucins in the intestine, promotes biofilm maturation and in virulent strains, it is a major determinant of infection, although how it carries out these functions is not well understood. Here we examine SslE from the E. coli Waksman and H10407 strains and using electron microscopy (EM), small angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) spectroscopy and biochemical analyses we show that SslE has a highly dynamic structure in solution. We also directly observe acidification within mature biofilms, describe a mechanism where SslE forms unique functional fibres under these conditions and determine that these SslE aggregates can bind cellulose, a major exopolysaccharide of many E. coli biofilms. Our data indicates that the spatial organization of SslE polymers and local pH are critical for biofilm maturation and SslE is a key factor that drives persistence of SslE-secreting bacteria during acidic stress.

Wagashi, also called Gassiréin the local Fulfulde language, is a traditional cheese produced from cow milk in Benin. For its preparation, milk is heated and coagulated with Calotropis procera extract. After coagulation, the heated curd obtained is drained, moulded and stained, or not, using Sorghum vulgare or Sorghum codatum panicles. It is the most commonly consumed dairy product as surrogate for meat or fish. In order to get better insights into the bacterial diversity of Wagashi, Illumina MiSeq amplicon sequencing targeting the V1-V2 region of the bacterial 16S rRNA gene was performed on two different Wagashi types (stained and not stained) from Benin. The results showed that the Lactobacillaceae (60%) and Streptococcaceae (38%), were the most abundant bacteria in the unstained cheese, whereas Streptococcaceae (89%) and Bacillaceae (10%) were the most prevalent in stained cheese. Moreover, at the species level, the microbial community structures of stained and unstained cheeses were significantly different. The main differentiating species were Lactobacillus bulgaricus, Streptococcus thermophilus, and Lactobacillus fermentum in unstained cheese, compared to Streptococcus infantarius, Aeromonas sp., Bacillus cereus, Bacillus subtilis and S. thermophilus in stained cheese. This work provides valuable insights into microbiology of cheese Wagashi and shows that the staining of this dairy product affects its bacterial composition. In particular, the presence of potential useful Lactobacilli and Streptococci in unstained cheese is to be further investigated for selecting functional bacteria for potential biotechnology applications. On the contrary, the presence of Bacilli in stained cheese needs additional study to detect potential foodborne strains or pathotypes.

Cells require thousands of unique proteins to be in the right place, at the right time, in the right amounts and with the right modifications. They do this through several processes collectively known as protein homeostasis. TORC1 is a principal regulator of protein homeostasis, coordinating protein synthesis and degradation. The proteasome, composed of a core particle and one or two regulatory particles, degrades unwanted protein. Diverse stresses cause a protein homeostasis imbalance: inhibiting TORC1 and the misfolded/damaged protein load. Following TORC1 inhibition, proteasome regulatory particle assembly chaperone (RPAC) translation is increased and thus cells assemble more proteasomes to degrade the damaged and misfolded proteins, thereby restoring protein homeostasis.

Using yeast, we identify an endocytic protein, Ede1, that interacts with and is critical for translation of RPAC mRNA following TORC1 inhibition. We find two further endocytic proteins important for RPAC translation regulation. Mutants of these proteins cause altered Arp2/3 activity, and hence altered formation of actin patches/endocytic sites. We show that RPAC mRNA is transported on actin cables and interacts with actin patches. TORC1 inhibition depolarises the actin cytoskeleton, causing RPAC mRNA accumulation on actin patches concurrent with translation. We demonstrate Ede1 is essential for RPAC mRNA localisation regulation following rapamycin treatment.

This work shows that, upon actin depolarisation, RPAC mRNA is recruited to actin patches, likely by Ede1, and translation occurs. Actin regulation is therefore a key element of proteasome (and therefore protein) homeostasis.

We present results demonstrating species-specificity and sub-species resolution by novel, automatically-designed metabarcoding primers for environmental DNA analysis.

Conventional metabarcoding remains a cornerstone of rapid, high-throughput environmental DNA (eDNA) community analysis and biodiversity assessment. Standard barcodes such as 16S (prokaryotes) and ITS1 (fungi/oomycetes) have been instrumental in identifying the complex composition of communities using total eDNA. However, standard barcodes have limitations in terms of resolution and quantitation and, though genus-level identification can be reliable, species-level identification is often not possible.

To overcome the limitations of resolution, we implemented extensions to the diagnostic primer design tool pdp (https://github.com/widdowquinn/find_differential_primers [https://github.com/widdowquinn/find_differential_primers]) that enable automated design of metabarcoding markers and corresponding primers that are (i) specific to a prescribed taxon at species level and (ii) capable of discriminating between members of the same species. This allows for rapid, high-throughput measurement of diversity below species level for a target organism.

We aimed to survey geographical distribution and pathogen transfer of the widespread plant pathogenic bacterium Pectobacterium atrosepticum (Pba). This organism has considerable sub-species taxonomic structure identifiable using MLST and with whole-genome methods, but which is not accessible using standard barcodes. We designed metabarcoding primers (202bp) specific to Pba using `pdp`, and established that these have resolution comparable to eight-gene MLST, revealing sub species-level diversity within single fields, and on the same individual plant host.

The Mycobacterium tuberculosiscomplex (MTBC) is a group of bacteria that show more than 99% genetic identity, yet they diverge in their host preference and the severity of associated disease. Mycobacterium bovis causes the disease in cattle and poses an economic challenge, with the cost of Irelands TB Eradication Scheme predicted to exceed €90 million in 2020.

The aim was to focus on the RD900 and pknH gene network in MTBC members. ThepknH gene encodes a transmembrane serine threonine protein kinase, that plays a role in the regulation of signalling pathways in the mycobacterial cell and has been linked to virulence. The RD900 or “region of difference 900” contains the tbD2 and pknH2 genes. The part of the genome that contains this region varies substantially across MTBC members.

The results of RNA-seq analysis showed that there are statistically significant levels of differential gene expression in wildtype and knock-in M. bovis strains.

The RD900 analysis across the MTBC showed that there is variation in this important genomic region. Analysis carried out by Mata et al. 2020, showed that this RD900 region had been independently lost in different MTBC lineages and strains. M. bovis has retained the RD900 region and this is believed to be linked to its increased virulence.

The majority of the M . africanum strains were found to possess the standard pknH, tbD2, pknH2, embR gene arrangement as is found in M. africanum GM041182, the strain that is typically used as a reference.

Based on work of Mata et al., 2020.

The amino sugar N-acetylglucosamine (GlcNAc), widely present in multiple cell-surfaces is efficiently utilized by human pathogen Candida albicans at the sites of infection as a survival strategy in different host niches. GlcNAc import inside the cell is mediated by GlcNAc transporter, Ngt1. In this present study, to investigate the Ngt1 dynamics, we have checked the sensitivity of Ngt1 for GlcNAc, expression kinetics and dynamics of Ngt1, stability of Ngt1 in the presence of unrelated carbon source like, glucose and endocytic trafficking of Ngt1. For this study we have used epi-fluorescence microscopy, Western blot and Northern blot analysis. We have observed that Ngt1 expression is prolific and highly sensitive to even minute amount (<2 μM) of GlcNAc. Ngt1 maintains its turnover in the plasma membrane by sugar stimulated endocytosis via UBI4 (polyubiquitin encoding locus) mediated ubiquitylation. Co-localization studies with different sub-cellular markers have revealed that Ngt1 follows a trafficking route via early endosome-late endosome-multi vesicular body (MVB)-Trans Golgi network for degradation. In conclusion, the study will provide a better understanding of endosomal trafficking for other membrane proteins in particular sugar transporters and also will open new therapeutic aspects in Candida albicans.

RND efflux pumps are important mediators of antibiotic resistance. RND efflux pumps including AcrB, are organized as tripartite systems, consisting of an inner membrane RND transporter, a periplasmic adaptor protein (PAP) and an outer membrane factor. We have previously identified the residues required for binding of the major PAP AcrA to the major RND pump AcrB and shown that there is promiscuity between the PAPs such that they can function with non-cognate pumps. AcrE is a PAP homologous to AcrA and AcrD and AcrF are RND pumps homologous to AcrB. This study aimed to determine whether the PAP AcrE can function with AcrD, which does not have its own PAP, to mediate efflux and whether the previously identified RND binding residues in AcrA and AcrE were also required for AcrD binding. The acrD and acrE genes were cloned into compatible vectors and co-transformed into a strain lacking acrAB, acrD and acrEF. When expressed together, acrDand acrE significantly decreased susceptibility of the efflux mutant strain to AcrD substrates including aztreonam, carbenicillin, cloxacillin, fusidic acid, nafcillin, novobiocin, oxacillin and ticarcillin, indicating that AcrE can also form a functional complex with AcrD. These experiments also highlighted that the substrate profile of AcrD in S. entericaand E. coli are different. Point mutations in the previously defined RND binding residues of AcrA and AcrE impaired AcrD-mediated efflux of substrate drugs which validates the interchangeability of AcrA and AcrE and highlights these residues as ideal drug targets for efflux inhibition to combat antimicrobial resistance.

Streptococcus pneumoniae is a major cause of global morbidity and mortality. It behaves as a commensal in the host nasopharynx, but can become pathogenic, invading the lungs, blood, and meninges. As such, identification of pneumococcal virulence and colonisation factors remains a major research objective. We previously described an experimental evolution approach for the identification of pneumococcal genes that make niche-specific contributions to fitness and virulence. Sequential passage of pneumococci through mouse models of nasopharyngeal-carriage and pneumonia was performed, generating bacterial lineages adapted to the nasopharynx and lungs, respectively.

Using RNA-Seq differential gene expression analysis, this study compared the transcriptomic profile of a nasopharynx-evolved pneumococcal lineage that showed evidence of enhanced nasopharyngeal colonisation potential, with a lab-adapted ancestor strain. Here, we describe how the genomic adaptations acquired by this lineage, and which we have demonstrated facilitate survival in the nasopharynx, can influence bacterial gene expression.

One key finding was the identification of five adjacent upregulated genes, representing a putative pneumococcal operon. These poorly characterised genes are predicted to encode a carbohydrate-scavenging pathway. Expression of the operon within the nasopharynx may facilitate sugar acquisition from host glycoproteins. Of note, the nasopharynx evolved pneumococcal lineage carries a single nucleotide insertion mutation immediately adjacent to the -10 element of the operon predicted promotor sequence. This may contribute to increased operon gene expression in the nasopharynx-adapted pneumococcus, thereby enhancing colonisation potential. Confirmatory mechanistic investigations are underway, which will aid the identification of pneumococcal virulence factors involved in the commensal to pathogen switch.