Casino, P. | Search bioRxiv

bioRxiv2025

Deciphering the role of histo-blood group antigens in bovine rotavirus C infection

Rotaviruses (RVs) are the main cause of viral diarrhea among infants, small children, and the young of many animal species. Histo-blood group antigens (HBGAs) are potential RV receptors and glycan composition on mucous surfaces influences host susceptibility and cross-species virus transmission. RVs exhibit genotype-dependent glycan binding and differences are due to sequence modifications in the VP8* domain of the spike protein VP4. Nevertheless, the molecular bases for this genotype-dependent glycan specificity, especially in non-A RVs, are not thoroughly understood. This study delves into how genotypic variations configure a novel binding site in the VP8* of a bovine P[3] rotavirus species C (RVC) strain to recognize H type-2 antigen (H2) and its precursor N-acetyl-lactosamine (LacNAc) using glycan binding assays, crystallography, and STD NMR. Results reveal a specific interaction of bovine P[3] RVC VP8* with H2 and LacNAc, more strongly with the latter. In the P[3] RVC VP8*-H2 interaction, the N-acetyl glucosamine moiety displays significant interaction, while galactose participates moderately and fucose binds weakly. Moreover, the bovine VP8* structure, resolved at 3 [A], shows specific structural features which differ from human RVC, as it contains two additional {beta}-strands ({beta}1 and {beta}2) contributing to {beta}-sheet2 and conformational changes that widens the cleft to allow different carbohydrate binding modes. These subtle changes in both sequence and structure explain the H2 precursor recognition, which is ubiquitous in human neonate intestine and in human and bovine milk, providing insights into P[3] RVC tropism and its potential zoonotic transmission. Author SummaryRotavirus C (RVC) represents an emerging pathogen with the ability to infect both humans and animals. It is widely acknowledged that host glycobiology plays a crucial role in determining susceptibility to RVs. Consequently, a better understanding of the interactions between RVs and carbohydrates is essential to know how the virus causes infection and to develop effective preventive strategies. Recent findings have unveiled the capability of the human RVC genotype P[2] to recognize the type A antigen through the VP8* spike protein although little is known about animal RVC strains. In this study, we describe the capacity of bovine P[3] RVC strain to bind H2 and LacNAc, both HBGAs. We also offer the structural explanation for the glycan differential recognition between human and bovine RVC strains. Our findings offer valuable insights into RVC attachment to host cells and its potential implications for species barriers.

microbiology↗

bioRxiv2022

Evolutionary analysis in Enterobacterales of the Rcs-repressor protein IgaA unveils two cytoplasmic small β-barrel domains central for function

The Rcs sensor system, comprised by the proteins RcsB/RcsC/RcsD and RcsF, is used by bacteria of the order Enterobacterales to withstand envelope damage. Under non-stress conditions, the system is repressed by the membrane protein IgaA. How IgaA has evolved within Enterobacterales in concert with the Rcs system has not been explored. Here, we report phylogenetic data supporting co-evolution of IgaA with the inner membrane proteins RcsC and RcsD. Functional assays showed that IgaA from representative genera as Shigella and Dickeya, but not those from Yersinia or the endosymbionts Photorhabdus and Sodalis, repress the Rcs system when expressed in a heterogenous host like Salmonella enterica serovar Typhimurium. IgaA structural features have therefore diverged among Enterobacterales. Modelling of IgaA structure unveiled one periplasmic and two cytoplasmic {beta}-rich architectures forming partially-closed small {beta}-barrel (SBB) domains related to OB (oligonucleotide/oligosaccharide binding motif) fold domains. Interactions among conserved residues were mapped in a connector linking SBB-1 domain of cytoplasmic region cyt1 to SBB-2 domain of region cyt2 (residues E180-R265); the C-terminus of cyt1 facing cyt2 (R188-E194-D309 and T191-H326); and, between cyt2-cyt3 regions (H293-E328-R686). These interactions identify a previously unnoticed "hybrid" SBB-2 domain. We also identified interactions absent in the IgaA variants not functional in S. Typhimurium, including H192-P249, which links cyt1 to cyt2, R255-D313 and D287-R314. A short -helix (6) located in the SSB-1 domain is also missing in the non-complementing IgaA tested. Taken together, our data support a central role of the two cytoplasmic SBB domains in IgaA function and evolution. SIGNIFICANCEThe "intracellular growth attenuator A" protein (IgaA) was first reported as repressor of the Rcs system in S. enterica serovar Typhimurium. IgaA orthologs were later studied in other genera and families of the Enterobacterales order, mainly in Escherichia coli. Despite intense investigation about the mechanism by which IgaA controls the Rcs system, the extent at which IgaA evolved within families of the Enterobacterales order has not been investigated. Using a combination of functional assays and in silico structural analyses, our work provides a detail map of conserved and divergent residues in IgaA representing interactions occurring in all Enterobacterales and others that may have diverged concomitantly to interacting proteins, probably for responding to specific environments. Future studies involving mutagenesis of these residues in IgaA of Enterobacterales families and genera of interest will certainly provide valuable insights into the regulation acting in the IgaA-Rcs axis.

microbiology↗