Biology subjects

Citti, C.

Publications and source records attributed to Citti, C..

8 recordsLinked to original sources

bioRxiv2026

Gut and nasal microbiome trajectories shape respiratory disease severity in calves exposed to antibiotic and pathogen challenge

Bovine respiratory disease complex (BRDC) is a multifactorial syndrome resulting from interactions among pathogens, host responses, and environmental stressors. However, substantial variation in disease severity persists among animals exposed to similar infectious challenges. To assess the contribution of the respiratory and gut microbiota to BRDC severity, we experimentally inoculated 28 calves with Mycoplasma bovis, bovine coronavirus (BCoV), and influenza D virus (IDV), with half receiving metaphylactic tilmicosin. Clinical trajectories were classified into mild, moderate, and severe phenotypes using unsupervised clustering of longitudinal health scores. Severe BRDC was associated with higher and more persistent nasal IDV loads, yet pathogen burden alone failed to explain the marked inter-individual variability in clinical outcomes. Tilmicosin-treated calves developed substantially milder disease despite comparable pathogen loads, coinciding with lower systemic inflammation, suggesting that host-pathogen interactions, rather than pathogen replication alone, influenced disease severity. Neither nasal nor faecal microbiota diversity consistently explained BRDC severity. However, calves that later developed severe disease displayed a more homogeneous nasal microbiota before infection, indicating that early microbial community structure may influence susceptibility. In contrast, the strongest disease-associated microbial signatures were observed in the faecal microbiota. Gut antibiotic-induced dysbiosis revealed associations between BRDC severity, specific intestinal bacterial taxa, systemic inflammation, and faecal short-chain fatty acid (SCFA) profiles. Notably, Chlamydia was strongly associated with disease severity, while several other taxa were linked to inflammatory biomarkers and SCFA concentrations. Collectively, these findings support a holobiont-driven model of BRDC in which disease severity emerges from interactions among pathogens, host inflammatory responses, and microbiota-associated functions. The stronger associations observed in the intestinal microbiota, along with links between gut bacteria, SCFAs, and disease outcomes, provide evidence for a functional gut-lung axis in calves and highlight microbial metabolism as a potential target for strategies to improve respiratory health and reduce antibiotic use.

microbiology↗

bioRxiv2026

Mucosal architecture, microbiota resilience and metabolic disruption reveal holobiont dynamics during bovine respiratory disease

The gut-airway axis is increasingly recognised as an important determinant of respiratory health in livestock. However, the interactions among respiratory infections, microbial communities, mucosal barrier function, and microbial metabolism that underpin this axis remain poorly understood. Here, we combined longitudinal immunological, mucosal, microbiological, and metabolic analyses to characterise host-microbiota interactions during antibiotic treatment and experimental bovine respiratory disease (BRD). We found that the bovine respiratory tract is organised as a hierarchically structured mucosal ecosystem, with compartment-specific mucin expression patterns associated with distinct microbial communities. Within this framework, increasing respiratory pathogen loads were associated with greater clinical severity, oxidative stress, acute inflammation, and targeted alterations in mucosal regulation and microbial composition, while serum amyloid A emerged as the host biomarker most closely reflecting infection intensity. Although infection altered host and microbial states, antimicrobial treatment represented the dominant ecological perturbation. Antibiotic exposure reshaped both respiratory and intestinal microbial communities, reducing diversity, increasing community drift, and driving coordinated ecological transitions across the gut-airway axis that exceeded the effects of infection alone. Importantly, these compositional changes were accompanied by profound reductions in acetate, propionate, butyrate, and valerate, identifying short-chain fatty acids as a major functional consequence of gut microbiota disruption. Integrative multi-omics analyses revealed that mucosal organisation, microbial diversity, microbial metabolism, and immune activity were embedded within a highly structured gut-airway network. Together, these findings support a holobiont view of BRD in which disease outcomes emerge not only from pathogen-host interactions but also from ecological disturbances that propagate across interconnected mucosal compartments. Notably, antimicrobial treatment affected ecosystem structure and function more than infection itself, highlighting potential trade-offs between pathogen control and microbiota stability. Preserving microbial resilience and its metabolic functions may therefore represent an important component of future strategies to improve respiratory health and disease resilience in cattle.

systems biology↗

bioRxiv2026

BoviScan: a Phage ImmunoPrecipitation Sequencing library to characterize the antibody response against Mycoplasma bovis.

Mycoplasma bovis is a major cattle pathogen and a global concern due to the limited efficient prophylactic tools and the emergence of antibiotic-resistant strains. Current control strategies rely primarily on surveillance, diagnostic and segregation or culling of infected animals. Despite substantial efforts to develop vaccines against M. bovis, no broadly effective solution is currently available. This situation is partly attributable to important gaps in our understanding of the host humoral immune response following infection, vaccination, or challenge. ELISA and Western blot assays have been widely used to assess M. bovis-specific antibody responses. While robust and easy to implement, these approaches are difficult to scale for hundreds of antigens in large animal cohorts and provide limited information on the specific antigens and epitopes targeted by the antibody response. To address these limitations, we developed BoviScan, a Phage Immunoprecipitation Sequencing (PhIP-Seq) library covering the pan-proteome of 295 M. bovis strains. We describe the design, synthesis, and validation of BoviScan using a panel of known-positive sera. We then applied the library to characterize antibody responses in a small cohort of experimentally immunized cattle and to monitor the longitudinal evolution of the humoral response in a single animal. Our results show that BoviScan enables high-throughput, high-resolution mapping of the antibody responses against M. bovis. This approach could be highly valuable for the study of host-pathogen interactions in mycoplasmas and the development of improved vaccines and serological assays.

microbiology↗

bioRxiv2025

Nucleoside Binding by a Surface Lipoprotein Governs Conjugative ICE Acquisition in Ruminant Mycoplasmas

Integrative and conjugative elements (ICEs) are major mediators of horizontal gene transfer (HGT) in bacteria. However, the role of recipient cells in their acquisition has received little attention. Using the ruminant pathogens Mycoplasma agalactiae and Mycoplasma bovis as minimal models, we combined genome-wide transposon mutagenesis with high-throughput mating assays to identify recipient factors required for ICE acquisition. The surface lipoprotein P48 emerged as the primary determinant of ICE uptake in both species. Structural and functional analyses revealed that P48 is the substrate-binding component of an ABC transporter with nucleoside-binding capacity. A single point mutation that abolished nucleoside binding drastically reduced ICE acquisition, demonstrating that P48-mediated nucleoside recognition is essential for conjugative transfer. However, ICE uptake did not require nucleoside transport, as inactivation of the transporter permease blocked nucleoside analog toxicity but not ICE invasion. Loss of P48 also triggered transcriptional activation of vestigial ICE genes, suggesting that surface recognition affects the intracellular state of the recipient. Remarkably, ICE transmission from recipient-derived donors was unaffected by P48 loss, underscoring its acquisition-specific role. Together, these results reveal a previously unrecognized, surface-exposed recipient factor critical for efficient ICE transfer in mycoplasmas and identify nucleotide binding as a central function in conjugation. By demonstrating that recipient-encoded functions can directly control ICE dissemination, this work challenges the donor-centric paradigm of bacterial conjugation and suggests new strategies to restrict horizontal gene flow in pathogenic and synthetic mycoplasmas. IMPORTANCEIntegrative and conjugative elements (ICEs) are mobile DNA elements that drive bacterial conjugation, a major process by which bacteria exchange genes. Although conjugation has been studied for decades, the focus has been almost exclusively on donor cells and the ICE itself, leaving the role of recipient cells largely overlooked. Using the wall-less ruminant pathogens Mycoplasma agalactiae and Mycoplasma bovis as minimal models, we discovered that a single recipient lipoprotein is required for efficient ICE uptake. Our data show that nucleoside recognition by P48, but not transport, is critical for conjugation, revealing an unexpected mechanistic link between nutrient sensing and gene acquisition. These findings shift the paradigm of conjugation from a donor-driven process to one jointly determined by donor and recipient functions. By identifying a recipient-encoded determinant of ICE transfer, this work opens new avenues to control horizontal gene flow in both pathogenic and engineered bacteria. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/677790v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@f7cc15org.highwire.dtl.DTLVardef@77a34eorg.highwire.dtl.DTLVardef@82699corg.highwire.dtl.DTLVardef@1e3be93_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Bacterial Conjugation in the Ruminant Pathogen Mycoplasma agalactiae is Influenced by Eukaryotic Host Factors

Bacterial conjugation plays a pivotal role in the evolution and adaptation of genome-reduced mycoplasmas. Despite their fast evolution rate, the conjugative properties of these organisms remain largely understudied, particularly in vivo. In the present study, the ruminant pathogen Mycoplasma agalactiae was used as a model organism to document the conjugative properties of mycoplasmas in environments of increasing complexity, from axenic to cell and organotypic culture conditions. Compared to axenic mating conditions, mycoplasma co-cultivation with goat epithelial cells or bovine precision-cut lung slices (PCLS) resulted in enhanced mating frequencies with high rates of M. agalactiae Integrative and Conjugative Element (ICEA) self-dissemination. These results were conditioned by the presence of eukaryotic cells in the culture and influenced by competition between mating partners but were not limited to M. agalactiae, as similar results were observed with Mycoplasma bovis. Mycoplasma conjugation ex vivo was further characterized by analyzing mycoplasma chromosomal transfer (MCT), a newly discovered mechanism of horizontal exchange of chromosomal DNA that generates mosaic genomes. Although closely associated with ICEA transfer, MCT was detected at low rates under cell and organotypic culture conditions suggesting a complex interplay between these two conjugative processes or a poor viability of the MCT progeny. Finally, mating experiments under nutrient-deprived conditions identified nucleotide stress as a potential factor influencing the modulation of mycoplasma conjugation by eukaryotic host cells. In conclusion, these results suggest that horizontal gene transfer in vivo is likely underestimated and provide valuable models to further studying mycoplasma conjugation ex vivo. IMPORTANCEConjugation is an evolutionary shortcut that bacteria use to exchange genetic information with their neighbors. Despite the fast evolution rate of the genome-reduced mycoplasmas, their conjugative properties remain largely understudied, particularly in vivo. Here we used the ruminant pathogen Mycoplasma agalactiae to study how mycoplasmas conjugate in co-culture with hosts-derived cells and tissues. Interestingly, conjugation was stimulated when mycoplasmas were co-cultured with eukaryotic cells. This was documented by monitoring the self-propagation of a mobile genetic element known as Integrative and Conjugative Element (ICE) and the exchange of chromosomal DNA leading to the formation of mosaic genomes. While ICE transfer was observed at high frequency, only a few mosaic genomes were detected in the presence of eukaryotic cells. Further data point towards nucleotide stress as a possible factor modulating mycoplasma conjugation in cellular environments. These results suggest that mycoplasma-host interactions may stimulate conjugation in vivo.

microbiology↗

bioRxiv2021

Genome mosaicism in field strains of Mycoplasma bovis as footprints of in-host horizontal chromosomal transfer

Horizontal gene transfer was long thought to be marginal in Mollicutes, but the capacity of some of these wall-less bacteria to exchange large chromosomal regions has been recently documented. Mycoplasma chromosomal transfer (MCT) is an unconventional mechanism that relies on the presence of a functional integrative conjugative element (ICE) in at least one partner and involves the horizontal acquisition of small and large chromosomal fragments from any part of the donor genome, which results in progenies composed of an infinitive variety of mosaic genomes. The present study focuses on Mycoplasma bovis, an important pathogen of cattle responsible for major economic losses worldwide. By combining phylogenetic tree reconstructions and detailed comparative genome analyses of 36 isolates collected in Spain (2016-2018) we confirmed the mosaic nature of 16 field isolates and mapped chromosomal transfers exchanged between their hypothetical ancestors. This study provides evidence that MCT can take place in the field, most likely during co-infections by multiple strains. Because mobile genetic elements (MGEs) are classical contributors of genome plasticity, the presence of phages, insertion sequences (ISs) and ICEs was also investigated. Data revealed that these elements are widespread within the M. bovis species and evidenced classical horizontal transfer of phages and ICEs in addition to MCT. These events contribute to wide-genome diversity and reorganization within this species and may have a tremendous impact on diagnostic and disease control. IMPORTANCEMycoplasma bovis is a major pathogen of cattle with significant detrimental economic and animal welfare on cattle rearing worldwide. Understanding the evolution and the adaptative potential of pathogenic mycoplasma species in the natural host is essential to combating them. In this study, we documented the occurrence of mycoplasma chromosomal transfer, an atypical mechanism of horizontal gene transfer, in field isolates of M. bovis that provide new insights into the evolution of this pathogenic species in their natural host. Despite these events are expected to occur at low frequency, their impact is accountable for genome-wide variety and reorganization within M. bovis species, which may compromise both diagnostic and disease control.

evolutionary biology↗

bioRxiv2021

Impacts of Mycoplasma agalactiae restriction-modification systems on pan-epigenome dynamics and genome plasticity

DNA methylation plays an important role in the biology of bacteria. Often associated with restriction modification (RM) systems, they also provide a defence against foreign DNA. Little is known regarding the methylome of the mycoplasma genus, which encompasses several pathogenic species with small genomes. Here, single molecule real-time (SMRT) and bisulphite sequencing combined with whole-genome analysis identified 19 methylated motifs associated with three orphan methyltransferases (MTases) and eight RM systems in Mycoplasma agalactiae, a ruminant pathogen and a model organism. All systems had a homolog in at least one phylogenetically distinct Mycoplasma spp. Our study also revealed that several superimposed genetic events may participate in the M. agalactiae dynamic epigenome landscape. These included (i) DNA shuffling and frameshift mutations that affect the MTase and restriction endonuclease content of a clonal population and (ii) gene duplication, erosion, and horizontal transfer that modulate MTase and RM repertoires of the species. Some of these systems were experimentally shown to play a major role in mycoplasma conjugative, horizontal DNA transfer. While the versatility of DNA methylation may contribute to regulating essential biological functions at cell and population levels, RM systems may be key in mycoplasma genome evolution and adaptation by controlling horizontal gene transfers.

microbiology↗

bioRxiv2018

The Integrative Conjugative Element (ICE) of Mycoplasma agalactiae: key elements involved in horizontal dissemination and influence of co-resident ICEs

The discovery of integrative conjugative elements (ICEs) in wall-less mycoplasmas and the demonstration of their role in massive gene flows within and across species has shed new light on the evolution of these minimal bacteria. Of these, ICEA of the ruminant pathogen Mycoplasma agalactiae represents a prototype and belongs to a new clade of the Mutatorlike superfamily that has no preferential insertion site and often occurs as multiple chromosomal copies. Here, functional genomics and mating experiments were combined to address ICEA functions and define the minimal ICEA chassis conferring conjugative properties to M. agalactiae. Data further indicated a complex interaction among co-resident ICEAs, since the minimal ICEA structure was influenced by the occurrence of additional ICEA copies that can trans-complement conjugative-deficient ICEAs. However, this cooperative behavior was limited to the CDS14 surface lipoprotein, which is constitutively expressed by co-resident ICEAs, and did not extend to other ICEA proteins including the cis-acting DDE recombinase and components of the mating channel whose expression was only sporadically detected. Remarkably, conjugative-deficient mutants containing a single ICEA copy knocked-out in cdsl4 can be complemented by neighboring cells expressing CDS14. This result, together with the conservation of CDS14 functions in closely related species, may suggest a way for mycoplasma ICEs to extend their interaction outside of their chromosomal environment. Overall, this study provides a first model of conjugative transfer in mycoplasmas and offers valuable insights towards the understanding of horizontal gene transfer in this highly adaptive and diverse group of minimal bacteria.\n\nIMPRTANCEIntegrative conjugative elements (ICEs) are self-transmissible mobile genetic elements that are key mediators of horizontal gene flow in bacteria. Recently, a new category of ICEs has been identified that confer conjugative properties to mycoplasmas, a highly adaptive and diverse group of wall-less bacteria with reduced genomes. Unlike classical ICEs, these mobile elements have no preferential insertion specificity and multiple mycoplasma ICE copies can be found randomly integrated into the host chromosome. Here, the prototype ICE of Mycoplasma agalactiae was used to define the minimal conjugative machinery and propose the first model of ICE transfer in mycoplasmas. This model unveils the complex interactions taking place among co-resident ICEs and suggests a way for these elements to extend their influence outside of their chromosomal environment. These data pave the way for future studies aiming at deciphering chromosomal transfer, an unconventional mechanism of DNA swapping that has been recently associated with mycoplasma ICEs.

microbiology↗