Covalent penicillin-protein conjugates elicit anti-drug antibodies that are clonally and functionally restricted.

Many archetypal and emerging classes of small-molecule therapeutics form covalent protein adducts. In vivo, both the resulting conjugates and their off-target side-conjugates have the potential to elicit antibodies, with implications for allergy and drug sequestration. Although ß-lactam antibiotics are a drug class long associated with these immunological phenomena, the molecular underpinnings of off-target drug-protein conjugation and consequent drug-specific immune responses remain incomplete. Here, using the classical ß-lactam penicillin G (PenG), we probe the B and T cell determinants of drug-specific IgG responses to such conjugates in mice. Deep B cell clonotyping reveals a dominant murine clonal antibody class encompassing phylogenetically-related IGHV1, IGHV5 and IGHV10 subgroup gene segments. Protein NMR and x-ray structural analyses reveal that these drive structurally convergent binding modes in adduct-specific antibody clones. Their common primary recognition mechanisms of the penicillin side-chain moiety (phenylacetamide in PenG)-regardless of CDRH3 length-limits cross-reactivity against other ß-lactam antibiotics. This immunogenetics-guided discovery of the limited binding solutions available to antibodies against side products of an archetypal covalent inhibitor now suggests future potential strategies for the 'germline-guided reverse engineering' of such drugs away from unwanted immune responses.

Epistasis, core-genome disharmony, and adaptation in recombining bacteria.

Recombination of short DNA fragments via horizontal gene transfer (HGT) can introduce beneficial alleles, create genomic disharmony through negative epistasis, and create adaptive gene combinations through positive epistasis. For non-core (accessory) genes, the negative epistatic cost is likely to be minimal because the incoming genes have not co-evolved with the recipient genome and are frequently observed as tightly linked cassettes with major effects. By contrast, interspecific recombination in the core genome is expected to be rare because disruptive allelic replacement is likely to introduce negative epistasis. Why then is homologous recombination common in the core of bacterial genomes? To understand this enigma, we take advantage of an exceptional model system, the common enteric pathogens Campylobacter jejuni and C. coli that are known for very high magnitude interspecies gene flow in the core genome. As expected, HGT does indeed disrupt co-adapted allele pairings, indirect evidence of negative epistasis. However, multiple HGT events enable recovery of the genome's co-adaption between introgressing alleles, even in core metabolism genes (e.g., formate dehydrogenase). These findings demonstrate that, even for complex traits, genetic coalitions can be decoupled, transferred, and independently reinstated in a new genetic background-facilitating transition between fitness peaks. In this example, the two-step recombinational process is associated with C. coli that are adapted to the agricultural niche.IMPORTANCEGenetic exchange among bacteria shapes the microbial world. From the acquisition of antimicrobial resistance genes to fundamental questions about the nature of bacterial species, this powerful evolutionary force has preoccupied scientists for decades. However, the mixing of genes between species rests on a paradox: 0n one hand, promoting adaptation by conferring novel functionality; on the other, potentially introducing disharmonious gene combinations (negative epistasis) that will be selected against. Taking an interdisciplinary approach to analyze natural populations of the enteric bacteria Campylobacter, an ideal example of long-range admixture, we demonstrate that genes can independently transfer across species boundaries and rejoin in functional networks in a recipient genome. The positive impact of two-gene interactions appears to be adaptive by expanding metabolic capacity and facilitating niche shifts through interspecific hybridization. This challenges conventional ideas and highlights the possibility of multiple-step evolution of multi-gene traits by interspecific introgression.

Loss of Pde1 function acts as an evolutionary gateway to penicillin resistance in Streptococcus pneumoniae.

Streptococcus pneumoniae is a major human pathogen and rising resistance to ß-lactam antibiotics, such as penicillin, is a significant threat to global public health. Mutations occurring in the penicillin-binding proteins (PBPs) can confer high-level penicillin resistance but other poorly understood genetic factors are also important. Here, we combined strictly controlled laboratory experiments and population analyses to identify a new penicillin resistance pathway that is independent of PBP modification. Initial laboratory selection experiments identified high-frequency pde1 mutations conferring S. pneumoniae penicillin resistance. The importance of variation at the pde1 locus was confirmed in natural and clinical populations in an analysis of >7,200 S. pneumoniae genomes. The pde1 mutations identified by these approaches reduce the hydrolytic activity of the Pde1 enzyme in bacterial cells and thereby elevate levels of cyclic-di-adenosine monophosphate and penicillin resistance. Our results reveal rapid de novo loss of function mutations in pde1 as an evolutionary gateway conferring low-level penicillin resistance. This relatively simple genomic change allows cells to persist in populations on an adaptive evolutionary pathway to acquire further genetic changes and high-level penicillin resistance.

Antibiotic-induced collateral damage to the microbiota and associated infections.

Antibiotics have transformed medicine, saving millions of lives since they were first used to treat a bacterial infection. However, antibiotics administered to target a specific pathogen can also cause collateral damage to the patient's resident microbial population. These drugs can suppress the growth of commensal species which provide protection against colonization by foreign pathogens, leading to an increased risk of subsequent infection. At the same time, a patient's microbiota can harbour potential pathogens and, hence, be a source of infection. Antibiotic-induced selection pressure can cause overgrowth of resistant pathogens pre-existing in the patient's microbiota, leading to hard-to-treat superinfections. In this Review, we explore our current understanding of how antibiotic therapy can facilitate subsequent infections due to both loss of colonization resistance and overgrowth of resistant microorganisms, and how these processes are often interlinked. We discuss both well-known and currently overlooked examples of antibiotic-associated infections at various body sites from various pathogens. Finally, we describe ongoing and new strategies to overcome the collateral damage caused by antibiotics and to limit the risk of antibiotic-associated infections.

Regulation of heterologous subtilin production in Bacillus subtilis W168.

BACKGROUND: Subtilin is a peptide antibiotic (lantibiotic) natively produced by Bacillus subtilis ATCC6633. It is encoded in a gene cluster spaBTCSIFEGRK (spa-locus) consisting of four transcriptional units: spaS (subtilin pre-peptide), spaBTC (modification and export), spaIFEG (immunity) and spaRK (regulation). Despite the pioneer understanding on subtilin biosynthesis, a robust platform to facilitate subtilin research and improve subtilin production is still a poorly explored spot. RESULTS: In this work, the intact spa-locus was successfully integrated into the chromosome of Bacillus subtilis W168, which is the by far best-characterized Gram-positive model organism with powerful genetics and many advantages in industrial use. Through systematic analysis of spa-promoter activities in B. subtilis W168 wild type and mutant strains, our work demonstrates that subtilin is basally expressed in B. subtilis W168, and the transition state regulator AbrB strongly represses subtilin biosynthesis in a growth phase-dependent manner. The deletion of AbrB remarkably enhanced subtilin gene expression, resulting in comparable yield of bioactive subtilin production as for B. subtilis ATCC6633. However, while in B. subtilis ATCC6633 AbrB regulates subtilin gene expression via SigH, which in turn activates spaRK, AbrB of B. subtilis W168 controls subtilin gene expression in SigH-independent manner, except for the regulation of spaBTC. Furthermore, the work shows that subtilin biosynthesis in B. subtilis W168 is regulated by the two-component regulatory system SpaRK and strictly relies on subtilin itself as inducer to fulfill the autoregulatory circuit. In addition, by incorporating the subtilin-producing system (spa-locus) and subtilin-reporting system (PpsdA-lux) together, we developed "online" reporter strains to efficiently monitor the dynamics of subtilin biosynthesis. CONCLUSIONS: Within this study, the model organism B. subtilis W168 was successfully established as a novel platform for subtilin biosynthesis and the underlying regulatory mechanism was comprehensively characterized. This work will not only facilitate genetic (engineering) studies on subtilin, but also pave the way for its industrial production. More broadly, this work will shed new light on the heterologous production of other lantibiotics.

Next-generation microbiology: from comparative genomics to gene function.

Microbiology is at a turning point in its 120-year history. Widespread next-generation sequencing has revealed genetic complexity among bacteria that could hardly have been imagined by pioneers such as Pasteur, Escherich and Koch. This data cascade brings enormous potential to improve our understanding of individual bacterial cells and the genetic basis of phenotype variation. However, this revolution in data science cannot replace established microbiology practices, presenting the challenge of how to integrate these new techniques. Contrasting comparative and functional genomic approaches, we evoke molecular microbiology theory and established practice to present a conceptual framework and practical roadmap for next-generation microbiology.

From Modules to Networks: a Systems-Level Analysis of the Bacitracin Stress Response in Bacillus subtilis.

Bacterial resistance against antibiotics often involves multiple mechanisms that are interconnected to ensure robust protection. So far, the knowledge about underlying regulatory features of those resistance networks is sparse, since they can hardly be determined by experimentation alone. Here, we present the first computational approach to elucidate the interplay between multiple resistance modules against a single antibiotic and how regulatory network structure allows the cell to respond to and compensate for perturbations of resistance. Based on the response of Bacillus subtilis toward the cell wall synthesis-inhibiting antibiotic bacitracin, we developed a mathematical model that comprehensively describes the protective effect of two well-studied resistance modules (BceAB and BcrC) on the progression of the lipid II cycle. By integrating experimental measurements of expression levels, the model accurately predicts the efficacy of bacitracin against the B. subtilis wild type as well as mutant strains lacking one or both of the resistance modules. Our study reveals that bacitracin-induced changes in the properties of the lipid II cycle itself control the interplay between the two resistance modules. In particular, variations in the concentrations of UPP, the lipid II cycle intermediate that is targeted by bacitracin, connect the effect of the BceAB transporter and the homeostatic response via BcrC to an overall resistance response. We propose that monitoring changes in pathway properties caused by a stressor allows the cell to fine-tune deployment of multiple resistance systems and may serve as a cost-beneficial strategy to control the overall response toward this stressor.IMPORTANCE Antibiotic resistance poses a major threat to global health, and systematic studies to understand the underlying resistance mechanisms are urgently needed. Although significant progress has been made in deciphering the mechanistic basis of individual resistance determinants, many bacterial species rely on the induction of a whole battery of resistance modules, and the complex regulatory networks controlling these modules in response to antibiotic stress are often poorly understood. In this work we combined experiments and theoretical modeling to decipher the resistance network of Bacillus subtilis against bacitracin, which inhibits cell wall biosynthesis in Gram-positive bacteria. We found a high level of cross-regulation between the two major resistance modules in response to bacitracin stress and quantified their effects on bacterial resistance. To rationalize our experimental data, we expanded a previously established computational model for the lipid II cycle through incorporating the quantitative action of the resistance modules. This led us to a systems-level description of the bacitracin stress response network that captures the complex interplay between resistance modules and the essential lipid II cycle of cell wall biosynthesis and accurately predicts the minimal inhibitory bacitracin concentration in all the studied mutants. With this, our study highlights how bacterial resistance emerges from an interlaced network of redundant homeostasis and stress response modules.

BceAB-Type Antibiotic Resistance Transporters Appear To Act by Target Protection of Cell Wall Synthesis.

Resistance against cell wall-active antimicrobial peptides in bacteria is often mediated by transporters. In low-GC-content Gram-positive bacteria, a common type of such transporters is BceAB-like systems, which frequently provide high-level resistance against peptide antibiotics that target intermediates of the lipid II cycle of cell wall synthesis. How a transporter can offer protection from drugs that are active on the cell surface, however, has presented researchers with a conundrum. Multiple theories have been discussed, ranging from removal of the peptides from the membrane and internalization of the drug for degradation to removal of the cellular target rather than the drug itself. To resolve this much-debated question, we here investigated the mode of action of the transporter BceAB of Bacillus subtilis We show that it does not inactivate or import its substrate antibiotic bacitracin. Moreover, we present evidence that the critical factor driving transport activity is not the drug itself but instead the concentration of drug-target complexes in the cell. Our results, together with previously reported findings, lead us to propose that BceAB-type transporters act by transiently freeing lipid II cycle intermediates from the inhibitory grip of antimicrobial peptides and thus provide resistance through target protection of cell wall synthesis. Target protection has so far only been reported for resistance against antibiotics with intracellular targets, such as the ribosome. However, this mechanism offers a plausible explanation for the use of transporters as resistance determinants against cell wall-active antibiotics in Gram-positive bacteria where cell wall synthesis lacks the additional protection of an outer membrane.

Adapting for life in the extreme.

Red algae have adapted to extreme environments by acquiring genes from bacteria and archaea.