Eukaryotic-like gephyrin and cognate membrane receptor coordinate corynebacterial cell division and polar elongation.

The order Corynebacteriales includes major industrial and pathogenic Actinobacteria such as Corynebacterium glutamicum or Mycobacterium tuberculosis. These bacteria have multi-layered cell walls composed of the mycolyl-arabinogalactan-peptidoglycan complex and a polar growth mode, thus requiring tight coordination between the septal divisome, organized around the tubulin-like protein FtsZ, and the polar elongasome, assembled around the coiled-coil protein Wag31. Here, using C. glutamicum, we report the discovery of two divisome members: a gephyrin-like repurposed molybdotransferase (Glp) and its membrane receptor (GlpR). Our results show how cell cycle progression requires interplay between Glp/GlpR, FtsZ and Wag31, showcasing a crucial crosstalk between the divisome and elongasome machineries that might be targeted for anti-mycobacterial drug discovery. Further, our work reveals that Corynebacteriales have evolved a protein scaffold to control cell division and morphogenesis, similar to the gephyrin/GlyR system that mediates synaptic signalling in higher eukaryotes through network organization of membrane receptors and the microtubule cytoskeleton.

High resolution cryo-EM and crystallographic snapshots of the actinobacterial two-in-one 2-oxoglutarate dehydrogenase.

Actinobacteria possess unique ways to regulate the oxoglutarate metabolic node. Contrary to most organisms in which three enzymes compose the 2-oxoglutarate dehydrogenase complex (ODH), actinobacteria rely on a two-in-one protein (OdhA) in which both the oxidative decarboxylation and succinyl transferase steps are carried out by the same polypeptide. Here we describe high-resolution cryo-EM and crystallographic snapshots of representative enzymes from Mycobacterium smegmatis and Corynebacterium glutamicum, showing that OdhA is an 800-kDa homohexamer that assembles into a three-blade propeller shape. The obligate trimeric and dimeric states of the acyltransferase and dehydrogenase domains, respectively, are critical for maintaining the overall assembly, where both domains interact via subtle readjustments of their interfaces. Complexes obtained with substrate analogues, reaction products and allosteric regulators illustrate how these domains operate. Furthermore, we provide additional insights into the phosphorylation-dependent regulation of this enzymatic machinery by the signalling protein OdhI.

Phages against Noncapsulated Klebsiella pneumoniae: Broader Host range, Slower Resistance.

Klebsiella pneumoniae (Kp), a human gut colonizer and opportunistic pathogen, is a major contributor to the global burden of antimicrobial resistance. Virulent bacteriophages represent promising agents for decolonization and therapy. However, the majority of anti-Kp phages that have been isolated thus far are highly specific to unique capsular types (anti-K phages), which is a major limitation to phage therapy prospects due to the highly polymorphic capsule of Kp. Here, we report on an original anti-Kp phage isolation strategy, using capsule-deficient Kp mutants as hosts (anti-Kd phages). We show that anti-Kd phages have a broad host range, as the majority are able to infect noncapsulated mutants of multiple genetic sublineages and O-types. Additionally, anti-Kd phages induce a lower rate of resistance emergence in vitro and provide increased killing efficiency when in combination with anti-K phages. In vivo, anti-Kd phages are able to replicate in mouse guts colonized with a capsulated Kp strain, suggesting the presence of noncapsulated Kp subpopulations. The original strategy proposed here represents a promising avenue that circumvents the Kp capsule host restriction barrier, offering promise for therapeutic development. IMPORTANCE Klebsiella pneumoniae (Kp) is an ecologically generalist bacterium as well as an opportunistic pathogen that is responsible for hospital-acquired infections and a major contributor to the global burden of antimicrobial resistance. In the last decades, limited advances have been made in the use of virulent phages as alternatives or complements to antibiotics that are used to treat Kp infections. This work demonstrates the potential value of an anti-Klebsiella phage isolation strategy that addresses the issue of the narrow host range of anti-K phages. Anti-Kd phages may be active in infection sites in which capsule expression is intermittent or repressed or in combination with anti-K phages, which often induce the loss of capsule in escape mutants.

FtsEX-independent control of RipA-mediated cell separation in Corynebacteriales.

The bacterial cell wall is a multi-layered mesh, whose major component is peptidoglycan (PG), a sugar polymer cross-linked by short peptide stems. During cell division, a careful balance of PG synthesis and degradation, precisely coordinated both in time and space, is necessary to prevent uncontrolled destruction of the cell wall. In Corynebacteriales, the D,L endopeptidase RipA has emerged as a major PG hydrolase for cell separation, and RipA defaults have major implications for virulence of the human pathogens Mycobacterium tuberculosis and Corynebacterium diphtheriae. However, the precise mechanisms by which RipA mediates cell separation remain elusive. Here we report phylogenetic, biochemical, and structural analysis of the Corynebacterium glutamicum homologue of RipA, Cg1735. The crystal structures of full-length Cg1735 in two different crystal forms revealed the C-terminal NlpC/P60 catalytic domain obtruded by its N-terminal conserved coiled-coil domain, which locks the enzyme in an autoinhibited state. We show that this autoinhibition is relieved by the extracellular core domain of the transmembrane septal protein Cg1604. The crystal structure of Cg1604 revealed a (ß/α) protein with an overall topology similar to that of receiver domains from response regulator proteins. The atomic model of the Cg1735-Cg1604 complex, based on bioinformatical and mutational analysis, indicates that a conserved, distal-membrane helical insertion in Cg1604 is responsible for Cg1735 activation. The reported data provide important insights into how intracellular cell division signal(s), yet to be identified, control PG hydrolysis during RipA-mediated cell separation in Corynebacteriales.

Ancient origin and constrained evolution of the division and cell wall gene cluster in Bacteria.

The division and cell wall (dcw) gene cluster in Bacteria comprises 17 genes encoding key steps in peptidoglycan synthesis and cytokinesis. To understand the origin and evolution of this cluster, we analysed its presence in over 1,000 bacterial genomes. We show that the dcw gene cluster is strikingly conserved in both gene content and gene order across all Bacteria and has undergone only a few rearrangements in some phyla, potentially linked to cell envelope specificities, but not directly to cell shape. A large concatenation of the 12 most conserved dcw cluster genes produced a robust tree of Bacteria that is largely consistent with recent phylogenies based on frequently used markers. Moreover, evolutionary divergence analyses show that the dcw gene cluster offers advantages in defining high-rank taxonomic boundaries and indicate at least two main phyla in the Candidate Phyla Radiation (CPR) matching a sharp dichotomy in dcw gene cluster arrangement. Our results place the origin of the dcw gene cluster in the Last Bacterial Common Ancestor and show that it has evolved vertically for billions of years, similar to major cellular machineries such as the ribosome. The strong phylogenetic signal, combined with conserved genomic synteny at large evolutionary distances, makes the dcw gene cluster a robust alternative set of markers to resolve the ever-growing tree of Bacteria.

SepF is the FtsZ anchor in archaea, with features of an ancestral cell division system.

Most archaea divide by binary fission using an FtsZ-based system similar to that of bacteria, but they lack many of the divisome components described in model bacterial organisms. Notably, among the multiple factors that tether FtsZ to the membrane during bacterial cell constriction, archaea only possess SepF-like homologs. Here, we combine structural, cellular, and evolutionary analyses to demonstrate that SepF is the FtsZ anchor in the human-associated archaeon Methanobrevibacter smithii. 3D super-resolution microscopy and quantitative analysis of immunolabeled cells show that SepF transiently co-localizes with FtsZ at the septum and possibly primes the future division plane. M. smithii SepF binds to membranes and to FtsZ, inducing filament bundling. High-resolution crystal structures of archaeal SepF alone and in complex with the FtsZ C-terminal domain (FtsZCTD) reveal that SepF forms a dimer with a homodimerization interface driving a binding mode that is different from that previously reported in bacteria. Phylogenetic analyses of SepF and FtsZ from bacteria and archaea indicate that the two proteins may date back to the Last Universal Common Ancestor (LUCA), and we speculate that the archaeal mode of SepF/FtsZ interaction might reflect an ancestral feature. Our results provide insights into the mechanisms of archaeal cell division and pave the way for a better understanding of the processes underlying the divide between the two prokaryotic domains.

Genome-wide analysis of the Firmicutes illuminates the diderm/monoderm transition.

The transition between cell envelopes with one membrane (Gram-positive or monoderm) and those with two membranes (Gram-negative or diderm) is a fundamental open question in the evolution of Bacteria. Evidence of the presence of two independent diderm lineages, the Halanaerobiales and the Negativicutes, within the classically monoderm Firmicutes has blurred the monoderm/diderm divide and specifically anticipated that other members with an outer membrane (OM) might exist in this phylum. Here, by screening 1,639 genomes of uncultured Firmicutes for signatures of an OM, we highlight a third and deep branching diderm clade, the Limnochordia, strengthening the hypothesis of a diderm ancestor and the occurrence of independent transitions leading to the monoderm phenotype. Phyletic patterns of over 176,000 protein families constituting the Firmicutes pan-proteome identify those that strongly correlate with the diderm phenotype and suggest the existence of new potential players in OM biogenesis. In contrast, we find practically no largely conserved core of monoderms, a fact possibly linked to different ways of adapting to repeated OM losses. Phylogenetic analysis of a concatenation of main OM components totalling nearly 2,000 amino acid positions illustrates the common origin and vertical evolution of most diderm bacterial envelopes. Finally, mapping the presence/absence of OM markers onto the tree of Bacteria shows the overwhelming presence of diderm phyla and the non-monophyly of monoderm ones, pointing to an early origin of two-membraned cells and the derived nature of the Gram-positive envelope following multiple OM losses.

One or two membranes? Diderm Firmicutes challenge the Gram-positive/Gram-negative divide.

How, when and why the transition between cell envelopes with one membrane (Gram-positives or monoderms) and two (Gram-negative or diderms) occurred in Bacteria is a key unanswered question in evolutionary biology. Different hypotheses have been put forward, suggesting that either the monoderm or the diderm phenotype is ancestral. The existence of diderm members in the classically monoderm Firmicutes challenges the Gram-positive/Gram-negative divide and provides a great opportunity to tackle the issue. In this review, we present current knowledge on the diversity of bacterial cell envelopes, including these atypical Firmicutes. We discuss how phylogenomic analysis supports the hypothesis that the diderm cell envelope architecture is an ancestral character in the Firmicutes, and that the monoderm phenotype in this phylum arose multiple times independently by loss of the outer membrane. Given the overwhelming distribution of diderm phenotypes with respect to monoderm ones, this scenario likely extends to the ancestor of all bacteria. Finally, we discuss the recent development of genetic tools for Veillonella parvula, a diderm Firmicute member of the human microbiome, which indicates it as an emerging new experimental model to investigate fundamental aspects of the diderm/monoderm transition.

Host nutritional status affects alphavirus virulence, transmission, and evolution.

Malnourishment, specifically overweight/obesity and undernourishment, affects more than 2.5 billion people worldwide, with the number affected ever-increasing. Concurrently, emerging viral diseases, particularly those that are mosquito-borne, have spread dramatically in the past several decades, culminating in outbreaks of several viruses worldwide. Both forms of malnourishment are known to lead to an aberrant immune response, which can worsen disease outcomes and reduce vaccination efficacy for viral pathogens such as influenza and measles. Given the increasing rates of malnutrition and spread of arthropod-borne viruses (arboviruses), there is an urgent need to understand the role of host nutrition on the infection, virulence, and transmission of these viruses. To address this gap in knowledge, we infected lean, obese, and undernourished mice with arthritogenic arboviruses from the genus Alphavirus and assessed morbidity, virus replication, transmission, and evolution. Obesity and undernourishment did not consistently influence virus replication in the blood of infected animals except for reductions in virus in obese mice late in infection. However, morbidity was increased in obese mice under all conditions. Using Mayaro virus (MAYV) as a model arthritogenic alphavirus, we determined that both obese and undernourished mice transmit virus less efficiently to mosquitoes than control (lean) mice. In addition, viral genetic diversity and replicative fitness were reduced in virus isolated from obese compared to lean controls. Taken together, nutrition appears to alter the course of alphavirus infection and should be considered as a critical environmental factor during outbreaks.

Chikungunya Virus Vaccine Candidates with Decreased Mutational Robustness Are Attenuated In Vivo and Have Compromised Transmissibility.

Chikungunya virus (CHIKV) is a reemerged arbovirus, a member of the Togaviridae family. It circulates through mosquito vectors mainly of the Aedes family and a mammalian host. CHIKV causes chikungunya fever, a mild to severe disease characterized by arthralgia, with some fatal outcomes described. In the past years, several outbreaks mainly caused by enhanced adaptation of the virus to the vector and ineffective control of the contacts between infected mosquito populations and the human host have been reported. Vaccines represent the best solution for the control of insect-borne viruses, including CHIKV, but are often unavailable. We designed live attenuated CHIKVs by applying a rational genomic design based on multiple replacements of synonymous codons. In doing so, the virus mutational robustness (capacity to maintain phenotype despite introduction of mutations to genotype) is decreased, driving the viral population toward deleterious evolutionary trajectories. When the candidate viruses were tested in the insect and mammalian hosts, we observed overall strong attenuation in both and greatly diminished signs of disease. Moreover, we found that the vaccine candidates elicited protective immunity related to the production of neutralizing antibodies after a single dose. During an experimental transmission cycle between mosquitoes and naive mice, vaccine candidates could be transmitted by mosquito bite, leading to asymptomatic infection in mice with compromised dissemination. Using deep-sequencing technology, we observed an increase in detrimental (stop) codons, which confirmed the effectiveness of this genomic design. Because the approach involves hundreds of synonymous modifications to the genome, the reversion risk is significantly reduced, rendering the viruses promising vaccine candidates.IMPORTANCE Chikungunya fever is a debilitating disease that causes severe pain to the joints, which can compromise the patient's lifestyle for several months and even in some grave cases lead to death. The etiological agent is chikungunya virus, an alphavirus transmitted by mosquito bite. Currently, there are no approved vaccines or treatments against the disease. In our research, we developed novel live attenuated vaccine candidates against chikungunya virus by applying an innovative genomic design. When tested in the insect and mammalian host, the vaccine candidates did not cause disease, elicited strong protection against further infection, and had low risk of reversion to pathogenic phenotypes.