Differential Use of the Two High-Oxygen-Affinity Terminal Oxidases of Brucella suis for In Vitro and Intramacrophagic Multiplication

Abstract

Expression of the high-oxygen-affinity cytochrome cbb3 and cytochrome bd ubiquinol oxidases of Brucella suis was studied in vitro and in the intramacrophagic niche, which was previously proposed to be oxygen limited. The cytochrome cbb3 oxidase was exclusively expressed in vitro, whereas the cytochrome bd oxidase was preferentially used inside macrophages and contributed to intracellular bacterial replication.Brucellosis, an anthropozoonosis encountered worldwide, is caused by the gram-negative intracellular pathogens Brucella spp., which utilize macrophages to multiply inside a specific niche (12) and to spread throughout the organism. Low levels of nutriments and oxygen, as revealed by the analysis of the intramacrophagic virulome (11), are major features of the Brucella suis replicative niche. Phagosomes of stimulated macrophages are known to have oxygen concentrations which are lower than those found in the extracellular environment (9). Furthermore, granulomatous structures generated by the immune system during localized infection within livers, spleens, or brains of patients are characterized by oxygen deficiency (2,20,21).Pathogenicity of brucellae and chronicity are due to the ability of the pathogen to adapt to the environmental conditions encountered in its replicative niche. To perform this task, Brucella has to modify its gene expression profile to rapidly adapt to the intracellular conditions. To this end, the bacteria induce a set of virulence genes, the main one being virB, encoding a type IV secretion system (17). Expression of genes involved in adaptation to oxygen-limited conditions appeared to be crucial for intramacrophagic survival of Brucella. A previous study identified a cydB mutant of Brucella abortus lacking the cytochrome bd oxidase with high affinity for oxygen as being highly attenuated in the mouse model of infection (6). Complete genome sequences (5,8,18) have revealed that Brucella possessed the locus ccoNOQP, potentially encoding another high-oxygen-affinity oxidase, the cytochrome cbb3-type terminal oxidase. We also identified a putative transcription regulator of the FixK/Fnr family in B. suis. The present study was undertaken to investigate (i) expression of the two operons encoding the cytochrome bd and cytochrome cbb3-type terminal oxidases in vitro as well as in bacteria obtained from infected cells and (ii) their respective roles in intracellular multiplication.

Different Roles of the Two High-Oxygen-Affinity Terminal Oxidases of Brucella suis : Cytochrome c Oxidase, but Not Ubiquinol Oxidase, Is Required for Persistence in Mice

“…The cbb3-type cytochrome c oxidase was specifically expressed in culture with maximal activation under microaerobiosis, whereas the cytochrome bd ubiquinol oxidase was preferentially expressed inside macrophages and involved in intracellular bacterial multiplication. These results indicated that Brucella needs a terminal oxidase with high affinity for oxygen for optimal survival inside the phagosomal compartment of the macrophage (18). In fact, the oxygen concentration inside phagosomes of stimulated macrophages is lower than that measured in the extracellular environment (11).…”

“…In a previous study, we showed that the two high-oxygen-affinity terminal oxidases of B. suis differently participated in adaptation to low-oxygen tension (18). The cbb3-type cytochrome c oxidase was specifically expressed in culture with maximal activation under microaerobiosis, whereas the cytochrome bd ubiquinol oxidase was preferentially expressed inside macrophages and involved in intracellular bacterial multiplication.…”

“…In order to evaluate a possible role of the cbb3-type and bd oxidases in virulence of B. suis, we examined the effect of mutations within the ccoNOQP and cydDCAB operons, respectively, on bacterial survival in mice. Wild-type B. suis 1330 and mutants harboring a deleted ccoN or cydB gene (18) were used to infect 7-week-old female BALB/c mice intraperitoneally at a dose of 10 5 CFU. At different time points, bacteria were recovered from the spleens and livers of five mice for each of the three strains.…”

The Escherichia coli CydX Protein Is a Member of the CydAB Cytochrome bd Oxidase Complex and Is Required for Cytochrome bd Oxidase Activity

“…Nitric oxide and reactive oxygen species are compounds released by macrophages to kill engulfed bacteria (20,21), and cytochrome bd oxidases have been shown to be less sensitive than other cytochrome oxidases to many such compounds (18,20). Consistent with cytochrome bd oxidase being important for bacterial survival within macrophages, cytochrome bd oxidase genes in Brucella suis, the cause of brucellosis, were shown to be induced upon macrophage engulfment, and were required for bacterial survival in the macrophage phagosomes (22). Together, these data suggest that cytochrome bd oxidases may play multiple roles in bacterial infection, helping bacteria survive periods of oxygen deprivation and exposure to cytotoxic agents released by the host immune system.…”

Stability of the cbb 3 -Type Cytochrome Oxidase Requires Specific CcoQ-CcoP Interactions

“…The same oxygen-dependent switch is observed in R. sphaeroides: aa 3 -Cox is the predominant oxidase under aerobic conditions, while cbb 3 -Cox is mainly expressed at low oxygen concentrations or during the transition from anaerobic to aerobic growth (1,24). Also, many pathogenic bacteria, such as Brucella suis (22) or Campylobacter jejuni (54), use cbb 3 -Cox mainly at low oxygen concentrations. On the other hand, in Thiobacillus denitrificans cbb 3 -Cox is highly upregulated under aerobic conditions (2).…”