Wolbachia are widespread bacterial symbionts of arthropods that manipulate host reproduction and can confer fitness benefits, such as protection against viral pathogens. Their self-spreading ability coupled with their antiviral effect has been harnessed in health programmes to curb the transmission of dengue virus by Aedes mosquitoes. In this context, a better understanding of evolutionary dynamics over short timescales is critical to inform and improve the efficacy of Wolbachia-based disease control interventions. Moreover, experimental evolution of Wolbachia may provide a powerful approach for generating novel symbiont strains with improved properties for disease control. Here we generated new mosquito cell lines by introducing five Wolbachia strains from Drosophila into the symbiont-free Aedes albopictus Aa23 cell line. Following transinfection, long-read whole-genome sequencing revealed multiple mutations, including structural changes, some of them being associated with major shifts in bacterial density. Signs of convergent evolution, an excess of non-synonymous mutations and comparisons with previous studies, indicate that Wolbachia evolution in cell culture is predominantly driven by selection and occurs faster than in insect hosts. Altogether, our study demonstrates that cell culture is a valuable tool to investigate short-term symbiont evolution, explore the genetic basis of bacterial density regulation and generate higher-density symbiont strains suitable for disease control.
As HIV-1 target cells, macrophages can be infected by cell-to-cell virus transfer from infected T cells through cell-cell fusion leading to the formation of virus-productive multinucleated giant cells (MGCs). We recently demonstrated that this route of infection escaped host cell restriction acting on the early steps of the viral life cycle. Here, we investigated whether restriction factors acting on late steps of HIV-1 replication were also bypassed in virus cell-to-cell infection of macrophages. We focused on the impact of the interferon(IFN)-induced factor BST-2/tetherin acting on virus release, a process counteracted by the Vpu viral protein. We show that deletion of Vpu did not significantly affect HIV-1 cell-to-cell infection of IFN-stimulated macrophages, and virus assembly in virus containing compartments was not affected in MGCs. Accordingly, depletion of BST-2 in IFN-stimulated macrophages had no impact on virus cell-to-cell transfer, formation of MGCs and virus production. However, cell-to-cell infection of macrophages was still sensitive to the nelfinavir protease inhibitor, whereas we previously showed that this route of infection was resistant to antiviral drugs acting on early steps of virus replication. Our results show that HIV-1 cell-to-cell infection of macrophages escapes restriction factors acting on both early and late steps of the virus life cycle, but displays resistance to antiviral drugs specifically acting on the early steps of virus replication.
Streptococcus suis (S. suis) is an encapsulated zoonotic pathogen that can form recalcitrant biofilms. Bacteriophage-derived endolysins and depolymerases represent promising antibiofilm agents, but currently, no report has described protein which displays both of lytic and capsule degradation activities simultaneously. Here, we mined the porcine gut virome and identified Lys33795, a dual-activity antibacterial protein that degrades both capsule and peptidoglycan of S. suis. Lys33795 consists of an N-terminal GH25_muramidase domain and a C-terminal cell wall binding domain (CBD). However, translational reinitiation at an internal start codon (M217) in the Lys33795 sequence generates an additional CBD that may assembles with the full-length protein to form an oligomeric complex. Interestingly, the GH25 muramidase truncation fragment of Lys33795 not only retains its classical function as a peptidoglycan hydrolase for degrading bacterial cell walls but also exhibits degradation activity towards capsule. This discovery contrasts sharply with the traditional functional understanding of the GH25 family. In addition, the CBD alone has a stable dimer and, as a nonstandard depolymerase without any classical polysaccharide-degrading catalytic domains, retains its degradation activity against S. suis capsule. We elucidated the molecular mechanism of Lys33795 exhibiting dual activity by combining mutagenesis, lysis, and capsule degradation activity assays. In addition, the results of in vitro and in vivo experiments showed that Lys33795 can effectively prevent and clear S. suis biofilms, and is more effective than single active mutants, and has a enhance effect when used in combination with antibiotics. This study describes the first multimeric bacteriophage protein with dual activities of lysis and capsule degradation against S. suis, encoded by a single gene. These findings provide valuable insights for designing potent antibacterial proteins targeting encapsulated pathogens.
Legionella pneumophila is the causative agent of Legionnaires’ disease, an accidental and severe form of pneumonia. The replication of L. pneumophila within alveolar macrophages requires the Dot/Icm type IV secretion system that translocates more than 330 bacterial effector proteins into the infected host cell. Many Dot/Icm effectors possess eukaryotic-like domains and manipulate host processes by functional mimicry. However, only a minority of effectors have been fully characterized. Redundancy among effector protein activities and host targets has complicated functional characterization of individual effector proteins. To explore the collective impact of Dot/Icm effectors on host cell processes, we created a library of 14 L. pneumophila genomic mutant strains lacking effector-rich regions (a total of 85 effector genes deleted) and used the multi-deletion mutants to interrogate host-pathogen interactions. While none of the mutants exhibited a defect in intracellular replication in THP-1 macrophages, several were attenuated in amoebae, including a mutant lacking a predicted amino acid transporter. In THP-1 cells, we observed that wild-type L. pneumophila 130b triggered a strong inflammatory and cellular stress transcriptional response in macrophages, whereas six mutants displayed reduced interferon signalling and one mutant displayed a marked decrease in the global expression of host heat shock response (HSR) genes. Through the creation of further sub-deletions, we found that the Dot/Icm effector kinase LegK4 was responsible for induction of the host HSR via the canonical transcription factor heat shock factor 1 (HSF1). Infection of THP-1 macrophages deficient in HSF1 revealed a potential role for the host HSR in the early stages of L. pneumophila infection. Together, these results reinforce the utility of multi-gene deletions to identify Dot/Icm effector phenotypes and the importance of the host HSR to L. pneumophila infection.
The intestinal microbiomes of men who have sex with men (MSM) are distinct as compared to non-MSM and may increase susceptibility to rectal HIV infection. We recently demonstrated that dysbiosis-associated disruptions to host antimicrobial immunity increase macaque susceptibility to rectal SIVmac239X acquisition. The described susceptibility-associated network identified Dual Oxidase 2 (DUOX2) – an epithelial sensor that catalyzes reactive oxygen species (ROS) production upon sensing pathobiont-derived uracil – as a likely contributor to increased SIV susceptibility. We hypothesized that intestinal, uracil bioavailability increases susceptibility to rectal HIV acquisition. Here, we treated rhesus macaques with oral uracil or uracil suppositories, prior to low-dose intra-rectal challenge with SIVmac239X. Oral treatment reduced pro-inflammatory cytokine production from peripheral T-cells but did not perturb rectal immunologic transcripts or epithelial ROS production and did not alter biomarkers of intestinal damage. Although treatment with uracil suppositories led to increased concentrations of fecal uracil and indices of intestinal damage, there were fewer immunological perturbations relative to oral treatment. Despite limited evidence of intestinal damage, oral uracil treatment increased susceptibility to intra-rectal SIV acquisition whereas uracil suppositories did not. These data provide mechanistic insights into intra-rectal SIV acquisition and may lead to novel therapeutic interventions that reduce incidence of HIV acquisition.
