The fast-growing Brucella suis Biovar 5 depends on phosphoenolpyruvate carboxykinase and pyruvate phosphate dikinase but not on Fbp and GlpX fructose-1, 6-bisphosphatases or isocitrate lyase for full virulence in laboratory models

Bacteria of the genus Brucella infect a range of vertebrates causing a worldwide extended zoonosis. The best-characterized brucellae infect domestic livestock, behaving as stealthy facultative intracellular parasites. This stealthiness depends on envelope molecules with reduced pathogen-associated molecular patterns, as revealed by the low lethality and ability to persist in mice of these bacteria. Infected cells are often engorged with brucellae without signs of distress, suggesting that stealthiness could also reflect an adaptation of the parasite metabolism to use local nutrients without harming the cell. To investigate this, we compared key metabolic abilities of Brucella abortus 2308 Wisconsin (2308W), a cattle biovar 1 virulent strain, and B. suis 513, the reference strain of the ancestral biovar 5 found in wild rodents. B. suis 513 used a larger number of C substrates and showed faster growth rates in vitro, two features similar to those of B. microti, a species phylogenomically close to B. suis biovar 5 that infects voles. However, whereas B. microti shows enhanced lethality and reduced persistence in mice, B. suis 513 was similar to B. abortus 2308W in this regard. Mutant analyses showed that B. suis 513 and B. abortus 2308W were similar in that both depend on phosphoenolpyruvate synthesis for virulence but not on the classical gluconeogenic fructose-1, 6-bisphosphatases Fbp-GlpX or on isocitrate lyase (AceA). However, B. suis 513 used pyruvate phosphate dikinase (PpdK) and phosphoenolpyruvate carboxykinase (PckA) for phosphoenolpyruvate synthesis in vitro while B. abortus 2308W used only PpdK. Moreover, whereas PpdK dysfunction causes attenuation of B. abortus 2308W in mice, in B. suis, 513 attenuation occurred only in the double PckA-PpdK mutant. Also contrary to what occurs in B. abortus 2308, a B. suis 513 malic enzyme (Mae) mutant was not attenuated, and this independence of Mae and the role of PpdK was confirmed by the lack of attenuation of a double Mae-PckA mutant. Altogether, these results decouple fast growth rates from enhanced mouse lethality in the brucellae and suggest that an Fbp-GlpX-independent gluconeogenic mechanism is ancestral in this group and show differences in central C metabolic steps that may reflect a progressive adaptation to intracellular growth

Dextran sulfate from Leuconostoc mesenteroides B512F exerts potent antiviral activity against SARS-CoV-2 in vitro and in vivo

The emergent human coronavirus SARS-CoV-2 and its resistance to current drugs makes the need for new potent treatments for COVID-19 patients strongly necessary. Dextran sulfate (DS) polysaccharides have long demonstrated antiviral activity against different enveloped viruses in vitro. However, their poor bioavailability has led to their abandonment as antiviral candidates. Here, we report for the first time the broad-spectrum antiviral activity of a DS-based extrapolymeric substance produced by the lactic acid bacterium Leuconostoc mesenteroides B512F. Time of addition assays with SARS-CoV-2 pseudoviruses in in vitro models confirm the inhibitory activity of DSs in the early stages of viral infection (viral entry). In addition, this exopolysaccharide substance also reports broad-spectrum antiviral activity against several enveloped viruses such as SARS-CoV-2, HCoV229E, HSV-1, in in vitro models and in human lung tissue. The toxicity and antiviral capacity of DS from L. mesenteroides was tested in vivo in mouse models which are susceptible to SARS-CoV-2 infection. The described DS, administered by inhalation, a new route of administration for these types of polymers, shows strong inhibition of SARS-CoV-2 infection in vivo, significantly reducing animal mortality and morbidity at non-toxic doses. Therefore, we suggest that it may be considered as a potential candidate for antiviral therapy against SARS-CoV-2Financial support for the study was provided by the REACT-EU 2021 grant from Comunidad de Madrid to the Project COVTRAVI19-CM, Plataformas y modelos preclínicos para el abordaje multidisciplinar en COVID-19 y en respuesta a futuras pandemia

Dextran sulfate from Leuconostoc mesenteroides B512F exerts potent antiviral activity against SARS-CoV-2 in vitro and in vivo

The emergent human coronavirus SARS-CoV-2 and its resistance to current drugs makes the need for new potent treatments for COVID-19 patients strongly necessary. Dextran sulfate (DS) polysaccharides have long demonstrated antiviral activity against different enveloped viruses in vitro. However, their poor bioavailability has led to their abandonment as antiviral candidates. Here, we report for the first time the broad-spectrum antiviral activity of a DS-based extrapolymeric substance produced by the lactic acid bacterium Leuconostoc mesenteroides B512F. Time of addition assays with SARS-CoV-2 pseudoviruses in in vitro models confirm the inhibitory activity of DSs in the early stages of viral infection (viral entry). In addition, this exopolysaccharide substance also reports broad-spectrum antiviral activity against several enveloped viruses such as SARS-CoV-2, HCoV229E, HSV-1, in in vitro models and in human lung tissue. The toxicity and antiviral capacity of DS from L. mesenteroides was tested in vivo in mouse models which are susceptible to SARS-CoV-2 infection. The described DS, administered by inhalation, a new route of administration for these types of polymers, shows strong inhibition of SARS-CoV-2 infection in vivo, significantly reducing animal mortality and morbidity at non-toxic doses. Therefore, we suggest that it may be considered as a potential candidate for antiviral therapy against SARS-CoV-2

"Phylogenomic insights into brucellaceae: The Pseudochrobactrum algeriensis case"

The genus Pseudochrobactrum encompasses free-living bacteria phylogenetically close to Ochrobactrum opportunistic pathogens and to Brucella, facultative intracellular parasites causing brucellosis, a worldwide-extended and grave zoonosis. Recently, Pseudochrobactrum strains were isolated from Brucella natural hosts on Brucella selective media, potentially causing diagnostic confusions. Strikingly, P. algeriensis was isolated from cattle lymph nodes, organs that are inimical to bacteria. Here, we analyse P. algeriensis potential virulence factors in comparison with Ochrobactrum and Brucella. Consistent with genomic analyses, Western-Blot analyses confirmed that P. algeriensis lacks the ability to synthesize the N-formylperosamine O-polysaccharide characteristic of the lipopolysaccharide (LPS) of smooth Brucella core species. However, unlike other Pseudochrobactrum but similar to some early diverging brucellae, P. algeriensis carries genes potentially synthetizing a rhamnose-based O-polysaccharide LPS. Lipid A analysis by MALDI-TOF demonstrated that P. algeriensis LPS bears a lipid A with a reduced pathogen-associated molecular pattern, a trait shared with Ochrobactrum and Brucella that is essential to generate a highly stable outer membrane and to delay immune activation. Also, although not able to multiply intracellularly in macrophages, the analysis of P. algeriensis cell lipid envelope revealed the presence of large amounts of cationic aminolipids, which may account for the extremely high resistance of P. algeriensis to bactericidal peptides and could favor colonization of mucosae and transient survival in Brucella hosts. However, two traits critical in Brucella pathogenicity are either significantly different (T4SS [VirB]) or absent (erythritol catabolic pathway) in P. algeriensis. This work shows that, while diverging in other characteristics, lipidic envelope features relevant in Brucella pathogenicity are conserved in Brucellaceae. The constant presence of these features strongly suggests that reinforcement of the envelope integrity as an adaptive advantage in soil was maintained in Brucella because of the similarity of some environmental challenges, such as the action of cationic peptide antibiotics and host defense peptides. This information adds knowledge about the evolution of Brucellaceae, and also underlines the taxonomical differences of the three genera compared

<i>Brucella</i> central carbon metabolism: an update

<p>The brucellae are facultative intracellular pathogens causing brucellosis, an important zoonosis. Here, we review the nutritional, genetic, proteomic and transcriptomic studies on <i>Brucella</i> carbon uptake and central metabolism, information that is needed for a better understanding of <i>Brucella</i> virulence. There is no uniform picture across species but the studies suggest primary and/or secondary transporters for unknown carbohydrates, lactate, glycerol phosphate, erythritol, xylose, ribose, glucose and glucose/galactose, and routes for their incorporation to central metabolism, including an erythritol pathway feeding the pentose phosphate cycle. Significantly, all brucellae lack phosphoenolpyruvate synthase and phosphofructokinase genes, which confirms previous evidence on glycolysis absence, but carry all Entner–Doudoroff (ED) pathway and Krebs cycle (and glyoxylate pathway) genes. However, glucose catabolism proceeds through the pentose phosphate cycle in the classical species, and the ED pathway operates in some rodent-associated brucellae, suggesting an ancestral character for this pathway in this group. Gluconeogenesis is functional but does not rely exclusively on classical fructose bisphosphatases. Evidence obtained using infection models is fragmentary but suggests the combined or sequential use of hexoses/pentoses, amino acids and gluconeogenic substrates. We also discuss the role of the phosphotransferase system, stringent reponse, quorum sensing, BvrR/S and sRNAs in metabolism control, an essential aspect of the life style of facultative intracellular parasites.</p

Brucella

The brucellae are facultative intracellular pathogens causing brucellosis, an important zoonosis. Here, we review the nutritional, genetic, proteomic and transcriptomic studies on Brucella carbon uptake and central metabolism, information that is needed for a better understanding of Brucella virulence. There is no uniform picture across species but the studies suggest primary and/or secondary transporters for unknown carbohydrates, lactate, glycerol phosphate, erythritol, xylose, ribose, glucose and glucose/galactose, and routes for their incorporation to central metabolism, including an erythritol pathway feeding the pentose phosphate cycle. Significantly, all brucellae lack phosphoenolpyruvate synthase and phosphofructokinase genes, which confirms previous evidence on glycolysis absence, but carry all Entner–Doudoroff (ED) pathway and Krebs cycle (and glyoxylate pathway) genes. However, glucose catabolism proceeds through the pentose phosphate cycle in the classical species, and the ED pathway operates in some rodent-associated brucellae, suggesting an ancestral character for this pathway in this group. Gluconeogenesis is functional but does not rely exclusively on classical fructose bisphosphatases. Evidence obtained using infection models is fragmentary but suggests the combined or sequential use of hexoses/pentoses, amino acids and gluconeogenic substrates. We also discuss the role of the phosphotransferase system, stringent reponse, quorum sensing, BvrR/S and sRNAs in metabolism control, an essential aspect of the life style of facultative intracellular parasites.</p

If you're not confused, you're not paying attention: Ochrobactrum is not Brucella

Bacteria of the genus Brucella are facultative intracellular parasites that cause brucellosis, a severe animal and human disease. Recently, a group of taxonomists merged the brucellae with the primarily free-living, phylogenetically related Ochrobactrum spp. in the genus Brucella. This change, founded only on global genomic analysis and the fortuitous isolation of some opportunistic Ochrobactrum spp. from medically compromised patients, has been automatically included in culture collections and databases. We argue that clinical and environmental microbiologists should not accept this nomenclature, and we advise against its use because (i) it was presented without in-depth phylogenetic analyses and did not consider alternative taxonomic solutions; (ii) it was launched without the input of experts in brucellosis or Ochrobactrum; (iii) it applies a non-consensus genus concept that disregards taxonomically relevant differences in structure, physiology, population structure, core-pangenome assemblies, genome structure, genomic traits, clinical features, treatment, prevention, diagnosis, genus description rules, and, above all, pathogenicity; and (iv) placing these two bacterial groups in the same genus creates risks for veterinarians, medical doctors, clinical laboratories, health authorities, and legislators who deal with brucellosis, a disease that is particularly relevant in low- and middle-income countries. Based on all this information, we urge microbiologists, bacterial collections, genomic databases, journals, and public health boards to keep the Brucella and Ochrobactrum genera separate to avoid further bewilderment and harm