Biology subjects

Johnston, P. R.

Publications and source records attributed to Johnston, P. R..

9 recordsLinked to original sources

bioRxiv2025

Clinical stringent response activation promotes conjugal transfer of staphylococcal resistance plasmids

Conjugative transfer of plasmids represents a major route through which antibiotic resistance genes are spread. In the case of the prevalent and deadly pathogen Staphylococcus aureus, >90% of clinical isolates carry at least one plasmid. While plasmid-encoded mechanisms (e.g. plasmid copy number) can influence conjugation frequency, host factors and environmental stimuli can also affect transmission. In particular, stress responses like the stringent response have been associated with increased movement of mobile genetic elements. We have previously shown that clinical mutations in the stringent response controller, Rel, lead to elevated levels of the alarmones (p)ppGpp and antibiotic tolerance in S. aureus. Here, we report that stringent response activation in these strains promotes the conjugal transfer of diverse staphylococcal resistance plasmids. We observed that clinical Rel mutations promote donation, but not receipt, of plasmids from the three families of staphylococcal plasmid and a mobilisable plasmid. This increased conjugation frequency could also be induced by chemical induction of the stringent response by mupirocin. Intriguingly, detailed experimental analysis revealed that the effect of elevated (p)ppGpp on plasmid donation was not due to CodY derepression, SOS response induction, increased plasmid copy number or increased expression of conjugation machinery genes. Further, transcriptomic analysis failed to identify any other putative plasmid- or host-derived mechanisms to explain this observation. Further investigations are required to explore the mechanistic link between the stringent response and conjugation, given the pervasive transcriptional and post-translational effects of (p)ppGpp. Overall, the association between Rel mutation and increased plasmid donation is alarming, especially as Rel mutations are being increasingly identified among clinical isolates.

microbiology↗

bioRxiv2024

Staphylococcus aureus COL: An Atypical Model Strain of MRSA that Exhibits Slow Growth and Antibiotic Tolerance

Methicillin-resistant Staphylococcus aureus (MRSA) has been a pathogen of global concern since its emergence in the 1960s. As one of the first MRSA strains isolated, COL has become a common model strain of S. aureus. Here we report that COL is, in fact, an atypical strain of MRSA that exhibits slow growth and multidrug tolerance. Genomic analysis identified three mutated genes in COL (rpoB, gltX and prs) with links to tolerance. Allele swapping experiments between COL and the closely related, non-tolerant Newman strain uncovered a complex interplay between these genes. However, Prs (phosphoribosyl pyrophosphate [PRPP] synthetase) accounted for most of the growth and tolerance phenotype of COL. Biochemical and transcriptomic analysis revealed that COL does not exhibit slow growth as a result of partial stringent response activation, as previously proposed. Instead, the COL Prs mutation greatly reduces the PRPP synthetase activity of the enzyme and leads to downregulation of pyrimidine, histidine and tryptophan synthesis, three pathways that rely on PRPP. Overall, our findings indicate that COL is an atypical, antibiotic-tolerant strain of MRSA whose isolation predates the previous first report of tolerance among clinical isolates. Characterisation of clinical Prs mutations and their relationship with tolerance requires further investigation.

microbiology↗

bioRxiv2023

Microbial Associates of the Elm Leaf Beetle: Uncovering the Absence of Resident Bacteria and the Influence of Fungi on Insect Performance

Microbial symbionts play crucial roles in the biology of many insects. While bacteria have been the primary focus of research on insect-microbe symbiosis, recent studies suggest that fungal symbionts may be just as important. The elm leaf beetle (ELB, Xanthogaleruca luteola) is a serious pest species of elm (Ulmus minor). Using culture-dependent and independent methods, we investigated the abundance and species richness of bacteria and fungi throughout various ELB life stages and generations, while concurrently analysing microbial communities on elm leaves. No persistent bacterial community was found to be associated with the ELB or elm leaves. By contrast, fungi were persistently present in the beetles feeding life stages and on elm leaves. Fungal community sequencing revealed a predominance of the genera Penicillium and Aspergillus in insects and on leaves. Culture-dependent surveys showed a high prevalence of two fungal colony morphotypes closely related to Penicillium lanosocoeruleum and Aspergillus flavus. Among these, the Penicillium morphotype was significantly more abundant on feeding-damaged compared to intact leaves, suggesting that the fungus thrives in the presence of the ELB. We assessed whether the detected prevalent fungal morphotypes influenced ELBs performance by rearing insects on i) surface-sterilised leaves, ii) leaves inoculated with Penicillium spores, and iii) leaves inoculated with Aspergillus spores. Insects feeding on Penicillium-inoculated leaves gained more biomass and tended to lay larger egg clutches than those consuming surface-sterilised leaves or Aspergillus-inoculated leaves. Our results demonstrate that the ELB does not harbour resident bacteria and that it might benefit from associating with Penicillium fungi. ImportanceOur study provides insights into the still understudied role of microbial symbionts in the biology of the ELB, a major pest of elms. Contrary to expectations, we found no persistent bacterial symbionts associated with the ELB or elm leaves. Our research thus contributes to the growing body of knowledge that not all insects rely on bacterial symbionts. While no persistent bacterial symbionts were detectable in the ELB and elm leaf samples, our analyses revealed the persistent presence of fungi, particularly Penicillium and Aspergillus on both elm leaves and in the feeding ELB stages. Moreover, when ELB were fed with fungus-treated elm leaves, we detected a potentially beneficial effect of Penicillium on the ELBs development and fecundity. Our results highlight the significance of fungal symbionts in the biology of this insect.

ecology↗

bioRxiv2022

IMMUNE GENE REGULATION IN THE GUT DURING METAMORPHOSIS IN TWO HOLO- VERSUS A HEMIMETABOLOUS INSECT

During complete metamorphosis, holometabolous insects remodel their entire anatomy, including the gut and must control their microbiota to avoid infectious disease. High activity of antimicrobial peptides and proteins in the gut during metamorphosis has been best described in several Lepidoptera. The immune system of the dipteran Drosophila melanogaster also controls the number of bacteria during metamorphosis. However, little is known about the regulation of immune genes during the larval-adult moult in Hemimetabola which undergo less drastic metamorphic changes. Different patterns of immune effector expression during metamorphosis were shown in a study comparing the lepidopteran Galleria mellonela and the orthopteran Gryllus bimaculatus, where G. mellonella showed a strong up-regulation of antimicrobial peptides in the gut at the larval-pupal moult but not G. bimaculatus. Whether these findings reflect general patterns within holometabolous versus hemimetabolous insects remains unclear. Using RNAseq, we compare the expression of immune effector genes in the gut during metamorphosis in two holometabolous (Calliphora vicina and Tenebrio molitor) and a hemimetabolous insect (Pyrrhocoris apterus). We found high read count abundances of differentially expressed immune effectors in the gut at the larval-pupal moult in C. vicina and T. molitor; no such high abundances were observed at the larval-adult moult in P. apterus. Our findings confirm that only complete metamorphosis elicits a prophylactic immune response as an adaptive response in holometabolous insects, which controls the microbiota during gut replacement.

evolutionary biology↗

bioRxiv2019

The effects of arbuscular mycorrhizal fungi (AMF) and Rhizophagus irregularis in soil microorganisms accessed by metatranscriptomics and metaproteomics

Arbuscular mycorrhizal fungi (AMF) form symbioses with approximately 80% of plant species and potentially benefit their hosts (e.g. nutrient acquisition) and the soil environment (e.g. soil aggregation). AMF also affect soil microbiota and soil multifunctionality. We manipulated AMF presence (via inoculation of non-sterile soil with Rhizophagus irregularis and using a hyphal compartment design) and used RNA-seq and metaproteomics to assess AMF roles in soil. The results indicated that AMF drove an active soil microbial community expressing transcripts and proteins related to nine metabolic functions, including the metabolism of C and N. We suggest two possible mechanisms: 1) the AMF hyphae produce exudates that select a beneficial community, or, 2) the hyphae compete with other soil microbes for available nutrients and consequently induce the community to mineralize nutrients from soil organic matter. We also identified candidate proteins that are potentially related to soil aggregation, such as Lpt and HSP60. Our results bridge microbial ecology and ecosystem functioning. We show that the AMF hyphosphere contains an active community related to soil respiration and nutrient cycling, thus potentially improving nutrient mineralization from soil organic matter and nutrient supply to the plants.

ecology↗

bioRxiv2019

Mating changes the genital microbiome in both sexes of the common bedbug Cimex lectularius across populations

Many bacteria live on host surfaces, in cells, and specific organ systems. Although gut microbiomes of many organisms are well-documented, the bacterial communities of reproductive organs, i.e. genital microbiomes, have received little attention. During mating, male and female genitalia interact and copulatory wounds can occur, providing an entrance for sexually transmitted microbes. Besides being potentially harmful to the host, invading microbes might interact with resident genital microbes and affect immunity. While sexual transmission of individual symbiont species is relatively well-understood, few studies have addressed how mating changes genital microbiomes. Here, we characterize male and female genital microbiomes in four different populations of the common bedbug Cimex lectularius and investigate mating-induced changes. We dissected reproductive organs from virgin and mated individuals to sequence their genital microbiomes. We show that mating changes genital microbiomes, suggesting bacteria are sexually transmitted. This raises the question how the hosts physiological and immunological mechanisms control mating-induced changes in their genital microbiomes. Also, genital microbiomes varied between populations and the sexes. This provides evidence for local and sex-specific adaptation of bacteria and hosts, again suggesting that bacteria might play an important role in shaping the evolution of reproductive traits. Coadaptation of genital microbiomes and reproductive traits might further lead to reproductive isolation between populations, giving reproductive ecology an important role in speciation. Future studies should investigate the transmission dynamics between the sexes and populations to uncover potential reproductive barriers.

evolutionary biology↗

bioRxiv2019

Bacteria primed by antimicrobial peptides develop tolerance and persist

Antimicrobial peptides (AMPs) are key components of innate immune defenses. Because of the antibiotic crisis, AMPs have also come into focus as new drugs. Here, we explore whether prior exposure to sublethal doses of AMPs increases bacterial survival and abets the evolution of resistance. We show that Escherichia coli primed by sublethal doses of AMPs develop tolerance and increase persistence by producing curli or colanic acid. We develop a population dynamic model that predicts that priming delays the clearance of infections and fuels the evolution of resistance. The effects we describe should apply to many AMPs and other drugs that target the cell surface. The optimal strategy to tackle tolerant or persistent cells requires high concentrations of AMPs and fast and long-lasting expression. Our findings also offer a new understanding of non-inherited drug resistance as an adaptive response and could lead to measures that slow the evolution of resistance.

evolutionary biology↗

bioRxiv2019

Eating in a losing cause: anorexia and macronutrient manipulation by a cockroach fails to boost immunity

BackgroundHost-pathogen interactions can lead to dramatic changes in host feeding behaviour. One aspect of this includes self-medication, where infected individuals consume substances such as toxins or alter their macronutrient consumption to enhance immune competence. Another widely adopted animal response to infection is illness-induced anorexia, which is thought to assist host immunity directly or by limiting the nutritional resources available to pathogens. Here, we recorded macronutrient preferences of the global pest cockroach, Blatta orientalis to investigate how shifts in host macronutrient dietary preference and quantity of carbohydrate (C) and protein (P) interact with immunity following bacterial infection. ResultsWe find that B. orientalis avoids diets enriched for P under normal conditions, and that high P diets reduce cockroach survival in the long term. However, following bacterial challenge, cockroaches significantly reduced their overall nutrient intake, particularly of carbohydrates, and increased the relative ratio of protein (P:C) consumed. Surprisingly, these behavioural shifts had a limited effect on cockroach immunity and survival, with minor changes to immune protein abundance and antimicrobial activity between individuals placed on different diets, regardless of infection status. ConclusionsWe show that cockroach feeding behaviour can be modulated by a pathogen, resulting in an illness-induced anorexia-like feeding response and a shift from a C-enriched to a more P:C equal diet. However, our results also indicate that such responses do not provide significant immune protection in B. orientalis, suggesting that the hosts dietary shift might also result from random rather than directed behaviour. The lack of an apparent benefit of the shift in feeding behaviour highlights a possible reduced importance for diet in immune regulation in these invasive animals, although further investigations employing pathogens with alternative infection strategies are warranted.

ecology↗

bioRxiv2019

Non-lethal oxidative stress boosts bacterial survival and evolvability under lethal exposure

Unicellular organisms have the prevalent challenge to survive under oxidative stress of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). ROS are present as by-products of photosynthesis and aerobic respiration. These reactive species are even employed by multicellular organisms as potent weapons against microbes. Although bacterial defences against lethal and sub-lethal oxidative stress have been studied in model bacteria, the role of fluctuating H2O2 concentrations remains unexplored. It is known that sub-lethal exposure of Escherichia coli to H2O2 results in enhanced survival upon subsequent exposure. Here we investigate the priming response to H2O2 at physiological concentrations. The basis and the duration of the response (memory) were also determined by time-lapse quantitative proteomics. We found that a low level of H2O2 induced several scavenging enzymes showing a long half-life, subsequently protecting cells from future exposure. We then asked if the phenotypic resistance against H2O2 alters the evolution of resistance against oxygen stress. Experimental evolution of H2O2 resistance revealed faster evolution and higher levels of resistance in primed cells. Several mutations were found to be associated with resistance in evolved populations affecting different loci but, counterintuitively, none of them was directly associated with scavenging systems. Our results have important implications for host colonisation and infections where microbes often encounter reactive oxygen species in gradients.

evolutionary biology↗