- Volume 168, Issue 12, 2022

Bacteria withstand antibiotic onslaughts by employing a variety of strategies, one of which is persistence. Persistence occurs in a bacterial population where a subpopulation of cells (persisters) survives antibiotic treatment and can regrow in a drug-free environment. Persisters may cause the recalcitrance of infectious diseases and can be a stepping stone to antibiotic resistance, so understanding persistence mechanisms is critical for therapeutic applications. However, current understanding of persistence is pervaded by paradoxes that stymie research progress, and many aspects of this cellular state remain elusive. In this review, we summarize the putative persister mechanisms, including toxin–antitoxin modules, quorum sensing, indole signalling and epigenetics, as well as the reasons behind the inconsistent body of evidence. We highlight present limitations in the field and underscore a clinical context that is frequently neglected, in the hope of supporting future researchers in examining clinically important persister mechanisms.

  Streptococcus pneumoniae   may inhabit the upper respiratory tract of humans without causing harm but it also causes diseases with high morbidity and mortality. It has excellent adaptive capabilities thanks to its ability to shuffle its genetic content by acquiring and incorporating DNA from other bacteria and is highly competent for genetic transformation. Sugar sensing, cleavage and transport ensure its fitness and survival in the host, and intracellular survival in macrophages has been linked to virulence. The polysaccharide capsule and toxin pneumolysin are the most important virulence determinants. Polysaccharide-based vaccines provide protection against the serotypes represented in vaccine formulations.

Daptomycin is a membrane-targeting lipopeptide antibiotic used in the treatment of infective endocarditis caused by multidrug-resistant Gram-positive bacteria such as Staphylococcus aureus , enterococci and viridans group streptococci. Despite demonstrating excellent in vitro activity and a low prevalence of resistant isolates, treatment failure is a significant concern, particularly for enterococcal infection. We have shown recently that human serum triggers daptomycin tolerance in S. aureus , but it was not clear if a similar phenotype occurred in other major infective endocarditis pathogens. We found that Enterococcus faecalis , Streptococcus gordonii or Streptococcus mutans grown under standard laboratory conditions were efficiently killed by daptomycin, whereas bacteria pre-incubated in human serum survived exposure to the antibiotic, with >99 % cells remaining viable. Incubation of enterococci or streptococci in serum led to peptidoglycan accumulation, as shown by increased incorporation of the fluorescent d-amino acid analogue HADA. Inhibition of peptidoglycan accumulation using the antibiotic fosfomycin resulted in a >tenfold reduction in serum-induced daptomycin tolerance, demonstrating the important contribution of the cell wall to the phenotype. We also identified a small contribution to daptomycin tolerance in E. faecalis from cardiolipin synthases, although this may reflect the inherent increased susceptibility of cardiolipin-deficient mutants. In summary, serum-induced daptomycin tolerance is a consistent phenomenon between Gram-positive infective endocarditis pathogens, but it may be mitigated using currently available antibiotic combination therapy.

Rhizobium adhering proteins or ‘Raps’ are secreted proteins identified in a very restricted group of rhizobial strains, specifically those belonging to R. leguminosarum and R. etli . The distinctive feature of members of the Rap family is the presence of one or two cadherin-like domains or CHDLs that are also present in numerous extracellular bacterial and archaeal proteins and were proposed to confer carbohydrate binding ability. We have previously made an in-depth characterization of RapA2, a calcium-binding lectin, composed by two CHDLs, involved in biofilm matrix remodelling in R. leguminosarum bv. viciae 3841. In this study, CHDLs derived from RapA2 were analysed in detail, finding significant structural and functional differences despite their considerable sequence similarity. Only the carboxy-terminal CHDL retained properties similar to those displayed by RapA2. Our findings were used to obtain a novel fluorescent probe to study biofilm matrix development by confocal laser scanning microscopy, and also to shed some light on the role of the ubiquitous CHDL domains in bacterial secreted proteins.

  Pseudomonas aeruginosa   uses quorum sensing (QS) to coordinate the expression of multiple genes necessary for establishing and maintaining infection. It has previously been shown that lasR QS mutations frequently arise in cystic fibrosis (CF) lung infections, however, there has been far less emphasis on determining whether other QS system mutations arise during infection or in other environments. To test this, we utilized 852 publicly available sequenced   P. aeruginosa   genomes from the   Pseudomonas   International Consortium Database (IPCD) to study   P. aeruginosa   QS mutational signatures. To study isolates by source, we focused on a subset of 654 isolates collected from CF, wounds, and non-infection environmental isolates, where we could clearly identify their source. We also worked with a small collection of isolates in vitro to determine the impact of lasR and pqs mutations on isolate phenotypes. We found that lasR mutations are common across all environments and are not specific to infection nor a particular infection type. We also found that the pqs system proteins PqsA, PqsH, PqsL and MexT, a protein of increasing importance to the QS field, are highly variable. Conversely, RsaL, a negative transcriptional regulator of the las system, was found to be highly conserved, suggesting selective pressure to repress las system activity. Overall, our findings suggest that QS mutations in   P. aeruginosa   are common and not limited to the las system; however, LasR is unique in the frequency of putative loss-of-function mutations.

There exists an enormous diversity of bacteria capable of human infection, but no up-to-date, publicly accessible list is available. Combining a pragmatic definition of pathogenicity with an extensive search strategy, we report 1513 bacterial pathogens known to infect humans described pre-2021. Of these, 73 % were regarded as established (have infected at least three persons in three or more references) and 27 % as putative (fewer than three known cases). Pathogen species belong to 10 phyla and 24 classes scattered throughout the bacterial phylogeny. We show that new human pathogens are discovered at a rapid rate. Finally, we discuss how our results could be expanded to a database, which could provide a useful resource for microbiologists. Our list is freely available and archived on GitHub and Zenodo and we have provided walkthroughs to facilitate access and use.

Peptide transporters play important nutritional and cell signalling roles in Bacillus subtilis, which are pronounced during stationary phase adaptations and development. Three high-affinity ATP-binding cassette (ABC) family transporters are involved in peptide uptake – the oligopeptide permease (Opp), another peptide permease (App) and a less well-characterized dipeptide permease (Dpp). Here we report crystal structures of the extracellular substrate binding proteins, OppA and DppE, which serve the Opp and Dpp systems, respectively. The structure of OppA was determined in complex with endogenous peptides, modelled as Ser-Asn-Ser-Ser, and with the sporulation-promoting peptide Ser-Arg-Asn-Val-Thr, which bind with K d values of 0.4 and 2 µM, respectively, as measured by isothermal titration calorimetry. Differential scanning fluorescence experiments with a wider panel of ligands showed that OppA has highest affinity for tetra- and penta-peptides. The structure of DppE revealed the unexpected presence of a murein tripeptide (MTP) ligand, l-Ala-d-Glu-meso-DAP, in the peptide binding groove. The mode of MTP binding in DppE is different to that observed in the murein peptide binding protein, MppA, from Escherichia coli , suggesting independent evolution of these proteins from an OppA-like precursor. The presence of MTP in DppE points to a role for Dpp in the uptake and recycling of cell wall peptides, a conclusion that is supported by analysis of the genomic context of dpp, which revealed adjacent genes encoding enzymes involved in muropeptide catabolism in a gene organization that is widely conserved in Firmicutes .

Despite its genome sequencing more than two decades ago, the majority of the genes of Mycobacterium tuberculosis remain functionally uncharacterized. Patatins are one such class of proteins that, despite undergoing an expansion in this pathogenic species compared to their non-pathogenic cousins, remain largely unstudied. Recent advances in protein structure prediction using machine learning tools such as AlphaFold2 have provided high-confidence predicted structures for all M. tuberculosis proteins. Here we present detailed analyses of the patatin family of M. tuberculosis using AlphaFold-predicted structures, providing insights into likely modes of regulation, membrane interaction and substrate binding. Regulatory domains within this family of proteins include cyclic nucleotide binding, lid-like domains and other helical domains. Using structural homologues, we identified the likely membrane localization mechanisms and substrate-binding sites. These analyses reveal diversity in their regulatory capacity, mechanisms of membrane binding and likely length of fatty acid substrates. Together, this analysis suggests unique roles for the eight predicted patatins of M. tuberculosis .