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The growing threat of bacterial resistance to antibiotics has led to the rise of anti-virulence strategies as a promising approach. These strategies aim to disarm bacterial pathogens and improve their clearance by the host immune system. Lipopolysaccharide, a key virulence factor in Gram-negative bacteria, has been identified as a potential target for anti-virulence agents. In this study, we focus on inhibiting HldA and HldE, bacterial enzymes from the heptose biosynthesis pathway, which plays a key role in lipopolysaccharide biosynthesis. We present the synthesis of two fluorinated non-hydrolysable heptose phosphate analogues. Additionally, the inhibitory activity of a family of eight heptose phosphate analogues against HldA and HldE was assessed. This evaluation revealed inhibitors with affinities in the low μM range, with the most potent compound showing inhibition constant values of 15.4 μM for HldA and 16.9 μM for HldE. The requirement for a phosphate group at the C-7 position was deemed essential for inhibitory activity, while the presence of a hydroxy anomeric group was found to be beneficial, a phenomenon rationalized through computational modeling. Additionally, the introduction of a single fluorine atom α to the phosphonate moiety conferred a slight advantage for inhibition. These findings suggest that mimicking the structure of d-glycero-β-d-manno-heptose 1,7-bisphosphate, the product of the phosphorylation step in heptose biosynthesis, could be a promising strategy to disrupt this biosynthetic pathway. In terms of the in vivo effects, these heptose phosphate analogues neither demonstrated significant LPS-disrupting effects nor exhibited growth inhibitory activity on their own. Additionally, they did not alter the susceptibility of bacteria to hydrophobic antibiotics. The highly charged nature of these molecules may hinder their ability to penetrate the bacterial cell wall. To overcome this limitation, alternative strategies such as incorporating protecting groups that facilitate their entry and can subsequently be cleaved within the bacterial cytoplasm could be explored., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Live attenuated vaccines (LAVs) whose virulence would be controlled at the tissue level could be a crucial tool to effectively fight intracellular bacterial pathogens, because they would optimize the induction of protective immune memory while avoiding the long-term persistence of vaccine strains in the host. Rational development of these new LAVs implies developing an exhaustive map of the bacterial virulence genes according to the host organs implicated. We report here the use of transposon sequencing to compare the bacterial genes involved in the multiplication of Brucella melitensis, a major causative agent of brucellosis, in the lungs and spleens of C57BL/6 infected mice. We found 257 and 135 genes predicted to be essential for B. melitensis multiplication in the spleen and lung, respectively, with 87 genes common to both organs. We selected genes whose deletion is predicted to produce moderate or severe attenuation in the spleen, the main known reservoir of Brucella, and compared deletion mutants for these genes for their ability to protect mice against challenge with a virulent strain of B. melitensis. The protective efficacy of a deletion mutant for the plsC gene, implicated in phospholipid biosynthesis, is similar to that of the reference Rev.1 vaccine but with a shorter persistence in the spleen. Our results demonstrate that B. melitensis faces different selective pressures depending on the organ and underscore the effectiveness of functional genome mapping for the design of new safer LAV candidates., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Barbieux et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The governing principles and suites of genes for lateral elongation or incorporation of new cell wall material along the length of a rod-shaped cell are well described. In contrast, relatively little is known about unipolar elongation or incorporation of peptidoglycan at one end of the rod. Recent work in three related model systems of unipolar growth (Agrobacterium tumefaciens, Brucella abortus, and Sinorhizobium meliloti) has clearly established that unipolar growth in the Hyphomicrobiales order relies on a set of genes distinct from the canonical elongasome. Polar incorporation of envelope components relies on homologous proteins shared by the Hyphomicrobiales, reviewed here. Ongoing and future work will reveal how unipolar growth is integrated into the alphaproteobacterial cell cycle and coordinated with other processes such as chromosome segregation and cell division., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Brucella abortus is a facultative, intracellular, zoonotic pathogen that resides inside macrophages during infection. This is a specialized niche where B. abortus encounters various stresses as it navigates through the macrophage. In order to survive this harsh environment, B. abortus utilizes post-transcriptional regulation of gene expression through the use of small regulatory RNAs (sRNAs). Here, we characterize a Brucella sRNAs called MavR (for MurF- and virulence-regulating sRNA), and we demonstrate that MavR is required for the full virulence of B. abortus in macrophages and in a mouse model of chronic infection. Transcriptomic and proteomic studies revealed that a major regulatory target of MavR is MurF. MurF is an essential protein that catalyzes the final cytoplasmic step in peptidoglycan (PG) synthesis; however, we did not detect any differences in the amount or chemical composition of PG in the ΔmavR mutant. A 6-nucleotide regulatory seed region within MavR was identified, and mutation of this seed region resulted in dysregulation of MurF production, as well as significant attenuation of infection in a mouse model. Overall, the present study underscores the importance of sRNA regulation in the physiology and virulence of Brucella., (© 2023 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)