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Journal articleHumphrey M, Larrouy-Maumus GJ, Furniss RCD, et al., 2021,
Colistin resistance in Escherichia coli confers protection of the cytoplasmic but not outer membrane from the polymyxin antibiotic
, MICROBIOLOGY-SGM, Vol: 167, Pages: 1-9, ISSN: 1350-0872Colistin is a polymyxin antibiotic of last resort for the treatment of infections caused by multi-drug-resistant Gram-negative bacteria. By targeting lipopolysaccharide (LPS), the antibiotic disrupts both the outer and cytoplasmic membranes, leading to bacterial death and lysis. Colistin resistance in Escherichia coli occurs via mutations in the chromosome or the acquisition of mobilized colistin-resistance (mcr) genes. Both these colistin-resistance mechanisms result in chemical modifications to the LPS, with positively charged moieties added at the cytoplasmic membrane before the LPS is transported to the outer membrane. We have previously shown that MCR-1-mediated LPS modification protects the cytoplasmic but not the outer membrane from damage caused by colistin, enabling bacterial survival. However, it remains unclear whether this observation extends to colistin resistance conferred by other mcr genes, or resistance due to chromosomal mutations. Using a panel of clinical E. coli that had acquired mcr −1, –1.5, −2, –3, −3.2 or −5, or had acquired polymyxin resistance independently of mcr genes, we found that almost all isolates were susceptible to colistin-mediated permeabilization of the outer, but not cytoplasmic, membrane. Furthermore, we showed that permeabilization of the outer membrane of colistin-resistant isolates by the polymyxin is in turn sufficient to sensitize bacteria to the antibiotic rifampicin, which normally cannot cross the LPS monolayer. These findings demonstrate that colistin resistance in these E. coli isolates is due to protection of the cytoplasmic but not outer membrane from colistin-mediated damage, regardless of the mechanism of resistance.
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Journal articlePathania M, Tosi T, Millership C, et al., 2021,
Structural basis for the inhibition of the Bacillus subtilis c-di-AMP cyclase CdaA by the phosphoglucomutase GlmM
, Journal of Biological Chemistry, Vol: 297, Pages: 1-15, ISSN: 0021-9258Cyclic-di-adenosine monophosphate (c-di-AMP) is an important nucleotide signaling molecule that plays a key role in osmotic regulation in bacteria. c-di-AMP is produced from two molecules of ATP by proteins containing a diadenylate cyclase (DAC) domain. In Bacillus subtilis, the main c-di-AMP cyclase, CdaA, is a membrane-linked cyclase with an N-terminal transmembrane domain followed by the cytoplasmic DAC domain. As both high and low levels of c-di-AMP have a negative impact on bacterial growth, the cellular levels of this signaling nucleotide are tightly regulated. Here we investigated how the activity of the B. subtilis CdaA is regulated by the phosphoglucomutase GlmM, which has been shown to interact with the c-di-AMP cyclase. Using the soluble B. subtilis CdaACD catalytic domain and purified full-length GlmM or the GlmMF369 variant lacking the C-terminal flexible domain 4, we show that the cyclase and phosphoglucomutase form a stable complex in vitro and that GlmM is a potent cyclase inhibitor. We determined the crystal structure of the individual B. subtilis CdaACD and GlmM homodimers and of the CdaACD:GlmMF369 complex. In the complex structure, a CdaACD dimer is bound to a GlmMF369 dimer in such a manner that GlmM blocks the oligomerization of CdaACD and formation of active head-to-head cyclase oligomers, thus suggesting a mechanism by which GlmM acts as a cyclase inhibitor. As the amino acids at the CdaACD:GlmM interphase are conserved, we propose that the observed mechanism of inhibition of CdaA by GlmM may also be conserved among Firmicutes.
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Journal articleMcKenna S, Giblin SP, Bunn RA, et al., 2021,
A highly efficient method for the production and purification of recombinant human CXCL8
, PLoS One, Vol: 16, Pages: 1-12, ISSN: 1932-6203Chemokines play diverse and fundamental roles in the immune system and human disease, which has prompted their structural and functional characterisation. Production of recombinant chemokines that are folded and bioactive is vital to their study but is limited by the stringent requirements of a native N-terminus for receptor activation and correct disulphide bonding required to stabilise the chemokine fold. Even when expressed as fusion proteins, overexpression of chemokines in E. coli tends to result in the formation of inclusion bodies, generating the additional steps of solubilisation and refolding. Here we present a novel method for producing soluble chemokines in relatively large amounts via a simple two-step purification procedure with no requirements for refolding. CXCL8 produced by this method has the correct chemokine fold as determined by NMR spectroscopy and in chemotaxis assays was indistinguishable from commercially available chemokines. We believe that this protocol significantly streamlines the generation of recombinant chemokines.
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Journal articlePruski P, Dos Santos Correia G, Lewis H, et al., 2021,
Direct on-swab metabolic profiling of vaginal microbiome host interactions during pregnancy and preterm birth
, Nature Communications, Vol: 12, ISSN: 2041-1723The pregnancy vaginal microbiome contributes to risk of preterm birth, the primary cause of death in children under 5 years of age. Here we describe direct on-swab metabolic profiling by Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) for sample preparation-free characterisation of the cervicovaginal metabolome in two independent pregnancy cohorts (VMET, n = 160; 455 swabs; VMET II, n = 205; 573 swabs). By integrating metataxonomics and immune profiling data from matched samples, we show that specific metabolome signatures can be used to robustly predict simultaneously both the composition of the vaginal microbiome and host inflammatory status. In these patients, vaginal microbiota instability and innate immune activation, as predicted using DESI-MS, associated with preterm birth, including in women receiving cervical cerclage for preterm birth prevention. These findings highlight direct on-swab metabolic profiling by DESI-MS as an innovative approach for preterm birth risk stratification through rapid assessment of vaginal microbiota-host dynamics.
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Journal articleHumphrey S, San Millan A, Toll-Riera M, et al., 2021,
Staphylococcal phages and pathogenicity islands drive plasmid evolution
, Nature Communications, Vol: 12, Pages: 1-15, ISSN: 2041-1723Conjugation has classically been considered the main mechanism driving plasmid transfer in nature. Yet bacteria frequently carry so-called non-transmissible plasmids, raising questions about how these plasmids spread. Interestingly, the size of many mobilizable and non transmissible plasmids coincides with the average size of phages (~40kb) or that of a family of pathogenicity islands, the phage-inducible chromosomal islands (PICIs, ~11 kb). Here, we show that phages and PICIs from Staphylococcus aureus can mediate intra- and inter-species plasmid transfer via generalised transduction, potentially contributing to non-transmissible plasmid spread in nature. Further, staphylococcal PICIs enhance plasmid packaging efficiency, and phages and PICIs exert selective pressures on plasmids via the physical capacity of their capsids, explaining the bimodal size distribution observed for non-conjugative plasmids. Our results highlight that transducing agents (phages, PICIs) have important roles in bacterial plasmid evolution and, potentially, in antimicrobial resistance transmission.
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Journal articleMullineaux Sanders C, Carson D, Hopkins E, et al., 2021,
Citrobacter amalonaticus inhibits the growth of Citrobacter rodentium in the gut lumen
, mBio, Vol: 5, Pages: 1-19, ISSN: 2150-7511The gut microbiota plays a crucial role in susceptibility to enteric pathogens, including Citrobacter rodentium, a model extracellular mouse pathogen that colonizes the colonic mucosa. C. rodentium infection outcomes vary between mouse strains, with C57BL/6 and C3H/HeN mice clearing or succumbing to the infection respectively. Kanamycin (Kan) treatment at the peak of C57BL/6 mouse infection with Kan-resistant C. rodentium resulted in re-localisation of the pathogen from the colonic mucosa and cecum to solely the cecal luminal contents; cessation of the Kan treatment resulted in rapid clearance of the pathogen. We now show that in C3H/HeN mice, following Kan-induced displacement of C. rodentium to the cecum, the pathogen stably colonizes the cecal lumen of 65% of the mice in the absence of continued antibiotic treatment, a phenomenon we term antibiotic-induced bacterial commensalisation (AIBC). AIBC C. rodentium was well-tolerated by the host, which showed little signs of inflammation; passaged AIBC C. rodentium robustly infected naïve C3H/HeN mice suggesting that the AIBC state is transient and did not select for genetically avirulent C. rodentium mutants. Following withdrawal of antibiotic treatment, 35% of C3H/HeN mice were able to prevent C. rodentium commensalisation in the gut lumen. These mice presented a bloom of a commensal species, Citrobacter amalonaticus, which inhibited the growth of C. rodentium in vitro in a contact-dependant manner, and luminal growth of AIBC C. rodentium in vivo. Overall our data suggest that commensal species can confer colonization resistance against closely-related pathogenic species.
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Journal articleLee WWY, Mattock J, Greig DR, et al., 2021,
Characterization of a pESI- like plasmid and analysis of multidrug-resistant Salmonella enterica Infantis isolates in England and Wales
, Microbial Genomics, Vol: 7, Pages: 1-11, ISSN: 2057-5858Salmonella enterica serovar Infantis is the fifth most common Salmonella serovar isolated in England and Wales. Epidemiological, genotyping and antimicrobial-resistance data for S. enterica Infantis isolates were used to analyse English and Welsh demographics over a 5 year period. Travel cases associated with S. enterica Infantis were mainly from Asia, followed by cases from Europe and North America. Since 2000, increasing numbers of S. enterica Infantis had multidrug resistance determinants harboured on a large plasmid termed ‘plasmid of emerging S. enterica Infantis’ (pESI). Between 2013 and 2018, 42 S. enterica Infantis isolates were isolated from humans and food that harboured resistance determinants to multiple antimicrobial classes present on a pESI-like plasmid, including extended-spectrum β-lactamases (ESBLs; blaCTX-M-65). Nanopore sequencing of an ESBL-producing human S. enterica Infantis isolate indicated the presence of two regions on an IncFIB pESI-like plasmid harbouring multiple resistance genes. Phylogenetic analysis of the English and Welsh S. enterica Infantis population indicated that the majority of multidrug-resistant isolates harbouring the pESI-like plasmid belonged to a single clade maintained within the population. The blaCTX-M-65 ESBL isolates first isolated in 2013 comprise a lineage within this clade, which was mainly associated with South America. Our data, therefore, show the emergence of a stable resistant clone that has been in circulation for some time in the human population in England and Wales, highlighting the necessity of monitoring resistance in this serovar.
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Journal articlePeriselneris J, Schelenz S, Loebinger M, et al., 2021,
Bronchiectasis severity correlates with outcome in patients with primary antibody deficiency
, THORAX, Vol: 76, Pages: 1036-1039, ISSN: 0040-6376- Author Web Link
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- Citations: 1
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Journal articlePanwar RB, Sequeira RP, Clarke TB, 2021,
Microbiota-mediated protection against antibiotic-resistant pathogens
, GENES AND IMMUNITY, Vol: 22, Pages: 255-267, ISSN: 1466-4879- Author Web Link
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- Citations: 12
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Journal articleHaag AF, Podkowik M, Ibarra-Chavez R, et al., 2021,
A regulatory cascade controls Staphylococcus aureus pathogenicity island activation
, Nature Microbiology, Vol: 6, Pages: 1300-1308, ISSN: 2058-5276Staphylococcal pathogenicity islands (SaPIs) are a family of closely related mobile chromosomal islands that encode and disseminate the superantigen toxins, toxic shock syndrome toxin 1 and superantigen enterotoxin B (SEB). They are regulated by master repressors, which are counteracted by helper phage–encoded proteins, thereby inducing their excision, replication, packaging and intercell transfer. SaPIs are major components of the staphylococcal mobilome, occupying five chromosomal att sites, with many strains harbouring two or more. As regulatory interactions between co-resident SaPIs could have profound effects on the spread of superantigen pathobiology, we initiated the current study to search for such interactions. Using classical genetics, we found that, with one exception, their regulatory systems do not cross-react. The exception was SaPI3, which was originally considered defective because it could not be mobilized by any known helper phage. We show here that SaPI3 has an atypical regulatory module and is induced not by a phage but by many other SaPIs, including SaPI2, SaPIbov1 and SaPIn1, each encoding a conserved protein, Sis, which counteracts the SaPI3 repressor, generating an intracellular regulatory cascade: the co-resident SaPI, when conventionally induced by a helper phage, expresses its sis gene which, in turn, induces SaPI3, enabling it to spread. Using bioinformatics analysis, we have identified more than 30 closely related coancestral SEB-encoding SaPI3 relatives occupying the same att site and controlled by a conserved regulatory module, immA–immR–str′. This module is functionally analogous but unrelated to the typical SaPI regulatory module, stl–str. As SaPIs are phage satellites, SaPI3 and its relatives are SaPI satellites.
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