Brucellosis is one of the most widespread bacterial zoonoses worldwide. Here, our aim was to identify the effector mechanisms controlling the early stages of intranasal infection with Brucella in C57BL/6 mice. During the first 48 hours of infection, alveolar macrophages (AMs) are the main cells infected in the lungs. Using RNA sequencing, we identified the aconitate decarboxylase 1 gene (Acod1; also known as Immune responsive gene 1), as one of the genes most upregulated in murine AMs in response to B. melitensis infection at 24 hours post-infection. Upregulation of Acod1 was confirmed by RT-qPCR in lungs infected with B. melitensis and B. abortus. We observed that Acod1-/- C57BL/6 mice display a higher bacterial load in their lungs than wild-type (wt) mice following B. melitensis or B. abortus infection, demonstrating that Acod1 participates in the control of pulmonary Brucella infection. The ACOD1 enzyme is mostly produced in mitochondria of macrophages, and converts cis-aconitate, a metabolite in the Krebs cycle, into itaconate. Dimethyl itaconate (DMI), a chemically-modified membrane permeable form of itaconate, has a dose-dependent inhibitory effect on Brucella growth in vitro. Interestingly, structural analysis suggests the binding of itaconate into the binding site of B. abortus isocitrate lyase. DMI does not inhibit multiplication of the isocitrate lyase deletion mutant ΔaceA B. abortus in vitro. Finally, we observed that, unlike the wt strain, the ΔaceA B. abortus strain multiplies similarly in wt and Acod1-/- C57BL/6 mice. These data suggest that bacterial isocitrate lyase might be a target of itaconate in AMs.

Capnocytophaga canimorsus is a dog oral commensal bacterium that causes rare but life-threatening generalized infections in humans who have been in contact with its animal hosts. Two other dog commensals, Capnocytophaga canis and Capnocytophaga cynodegmi, cause rare, mild local infections. To date, nine capsular serovars have been described in C. canimorsus. Here, we serotyped 112 strains of Capnocytophaga spp. isolated from human infections. The C. canimorsus strains (86 of 96, 89.6%) belonged to serovars A, B, or C with relative frequencies of approximately 30% for each serovar. The high prevalence of the A, B, and C serovars in strains isolated from humans, compared to the previously described low prevalence of these serovars among dog isolates (7.6%), confirms that these three serovars are more virulent to humans than other serovars and suggests that the low incidence of disease may be linked to the low prevalence of the A, B, and C serovars in dogs. We serotyped six strains of C. canis and ten strains of C. cynodegmi and, surprisingly, found one C. canis and three C. cynodegmi strains to be of capsular serovar B. This observation prompted us to test 34 dog-isolated C. canis and 16 dog-isolated C. cynodegmi strains. We found four C. canis strains belonging to serovar A and one belonging to serovar F. In contrast, no dog-isolated C. cynodegmi strain could be typed with the available antisera. This work demonstrates that virulence-associated capsular polysaccharides (A, B, and C) are not specific to the C. canimorsus species.

We assembled a collection of 73 Capnocytophaga canimorsus isolates obtained from blood cultures taken from patients treated at Helsinki University Hospital (Helsinki, Finland) during 2000–2017. We serotyped these isolates by PCR and Western blot and attempted to correlate pathogen serovar with patient characteristics. Our analyses showed, in agreement with previous research, that 3 C. canimorsus serovars (A–C) caused most (91.8%) human infections, despite constituting only 7.6% of isolates found in dogs. The 3 fatalities that occurred in our cohort were equally represented by these serovars. We found 2 untypeable isolates, which we designated serovars J and K. We did not detect an association between serovar and disease severity, immune status, alcohol abuse, or smoking status, but dog bites occurred more frequently among patients infected with non-A–C serovars. Future research is needed to confirm serovar virulence and develop strategies to reduce risk for these infections in humans.

Brucellaceae are facultative intracellular Gram-negative cocobacilli, belonging to the Rhizobiales order of α-Proteobacteria family.Members of the Brucella genus are responsible for a worldwide zoonosis known as brucellosis, with important consequences for health and economy. In animal livestock, the disease leads to abortion and sterility. Human brucellosis, mainly caused by B. melitensis, B. suis, B. abortus, and B. canis, is characterized by an acute phase with periodic undulant fever. The illness can evolve to a chronic infection, which could also be associated with endocarditis or meningitis (Pappas et al., 2005).In vitro HeLa and macrophage cell infection by B. abortus follows a three-step model in which bacteria are found in three successive types of vacuoles (Celli, 2019). In Hela cells and RAW264.7 macrophages-like cells, G1 phase bacteria ("new-born") enter the cells, reside in an endosomal Brucella containing vacuole (eBCV) but remain blocked in G1 during the first hours after invasion. B. abortus resumes growth before the eBCV reaches the replicative niche in the endoplasmic reticulum (rBCV) (Deghelt et al., 2014). After extensive replication in the rBCV, bacteria are captured in autophagosome-like compartments forming an autophagic BCV (aBCV) (Celli, 2019).One characteristic of Brucellae shared with Rhizobiales, is their asymmetric unipolar growth, as it occurs at one pole during elongation and at midcell during the cell division (Brown et al., 2012).Recently, it has been shown that newly synthesized peptidoglycan (PG) and lipopolysaccharide are only inserted at one pole of the bacteria, named the growing pole (Vassen et al., 2019). The growing pole

Lipopolysaccharide is essential for most Gram-negative bacteria as it is a main component of the outer membrane. In the pathogen Brucella abortus, smooth lipopolysaccharide containing the O-antigen is required for virulence. Being part of the Rhizobiales, Brucella spp. display unipolar growth and lipopolysaccharide was shown to be incorporated at the active growth sites, i.e. the new pole and the division site. By localizing proteins involved in the lipopolysaccharide transport across the cell envelope, from the inner to the outer membrane, we show that the lipopolysaccharide incorporation sites are determined by the inner membrane complex of the lipopolysaccharide transport system. Moreover, we identify the main O-antigen ligase of Brucella spp. involved in smooth lipopolysaccharide synthesis. Altogether, our data highlight a layer of spatiotemporal organization of the lipopolysaccharide biosynthesis pathway and identify an original class of bifunctional O-antigen ligases.