Brucella melitensis Wzm/Wzt System: Changes in the Bacterial Envelope Lead to Improved Rev1Δwzm Vaccine Properties

Abstract

The lipopolysaccharide (LPS) O-polysaccharide (O-PS) is the main virulence factor in Brucella. After synthesis in the cytoplasmic membrane, O-PS is exported to the periplasm by the Wzm/Wzt system, where it is assembled into a LPS. This translocation also engages a bactoprenol carrier required for further biosynthesis pathways, such as cell wall biogenesis. Targeting O-PS export by blockage holds great potential for vaccine development, but little is known about the biological implications of each Wzm/Wzt moiety. To improve this knowledge and to elucidate its potential application as a vaccine, we constructed and studied wzm/wzt single- and double-deletion mutants, using the attenuated strain Brucella melitensis Rev1 as the parental strain. This allowed us to describe the composition of Brucella peptidoglycan for the first time. We observed that these mutants lack external O-PS yet trigger changes in genetic transcription and in phenotypic properties associated with the outer membrane and cell wall. Both mutants are highly attenuated; unexpectedly, Rev1Δwzm also excels as an immunogenic and effective vaccine against B. melitensis and Brucella ovis in mice, revealing that low persistence is not at odds with efficacy. Rev1Δwzm is attenuated in BeWo trophoblasts, does not infect mouse placentas, and is safe in pregnant ewes. Overall, these attributes and the minimal serological interference induced in sheep make Rev1Δwzm a highly promising vaccine candidate.

Bru SIC: a novel selective medium for the primary isolation of Brucella in veterinary samples

“…Before starting safety experiments on Brucella infection in the natural host, the suitability of the selective culture media must be assessed to avoid biased results, such as false negatives or misclassified levels of infection. Accordingly, the initial aim of this study was to evaluate the growth of two B. melitensis R-LPS mutants in the recommended selective culture media FM and CM ( 1 ) due to their high susceptibility to polymyxins B and E ( 27 , 28 ), which are present in FM and CM, respectively. As hypothesized, both FM and CM significantly inhibited the growth of both B. melitensis rough mutants, particularly Rev1Δ wzm .…”

“…To determine the inhibition induced by the antibiotics in CM, individually and combined, we used the standard microdilution broth method, as described previously ( 27 , 54 ), and chequerboard titration ( 55 ). Briefly, 2-fold serial dilutions of vancomycin (0.625 to 640 μg/mL), colistin (0.25 to 750 μg/mL), and nitrofurantoin (0.625 to 320 μg/mL) were prepared in cation-adjusted Müller-Hinton medium (M-H; BD, Heidelberg, Germany), immediately dispensed (100 μL/well) in 96-well polystyrene plates (Sarstedt, Nümbrecht, Germany) and mixed with an equal volume of bacterial suspension adjusted to optical density at 750 nm of 0.109 (≈1.5 × 10 9 CFU/mL) and diluted 1:100 in M-H. Each concentration was analyzed in duplicate, and plain M-H wells with or without bacteria were used as controls.…”

“…The performance of BSM for the primary isolation of Rev1Δ wzm and 16MΔ wzm mutants was determined by duplicate culturing (at 37°C for 14 days) of 1,502 samples from 141 experimentally infected sheep; CM and BAB were used as controls. The cultured samples were swabs impregnated in vaginal fluid, semen, milk, amniotic fluid, cotyledons, or fetuses, as well as in liver, spleen, seminal vesicle, epididymis, uterus, mammary gland, and lymph node tissues, all of which were obtained, processed, and homogenated in PBS as previously described ( 27 ). The identity of the presumptive 16MΔ wzm and Rev1Δ wzm colonies was confirmed by subculturing them in BAB, and further analysis was carried out to differentiate Rev1 from 16M and to detect the deletion in wzm by PCR-RFLP and PCR-WZM, as previously described ( 27 , 56 ).…”

“…The cultured samples were swabs impregnated in vaginal fluid, semen, milk, amniotic fluid, cotyledons, or fetuses, as well as in liver, spleen, seminal vesicle, epididymis, uterus, mammary gland, and lymph node tissues, all of which were obtained, processed, and homogenated in PBS as previously described ( 27 ). The identity of the presumptive 16MΔ wzm and Rev1Δ wzm colonies was confirmed by subculturing them in BAB, and further analysis was carried out to differentiate Rev1 from 16M and to detect the deletion in wzm by PCR-RFLP and PCR-WZM, as previously described ( 27 , 56 ). The number of CFU/plate was used to determine the percentage of increasing productivity as follows: [(CFU in medium A − CFU in medium B)/CFU in medium B] × 100.…”

“…Nevertheless, susceptibility to polymyxins B (in FM) or E (colistin in CM) has been widely associated with attenuated Brucella mutants with exposed anionic phosphate groups of the lipid A lipopolysaccharide (LPS), such as rough LPS (R-LPS) mutants ( 20 – 28 ). In our laboratory, PCR overlapping was used to obtain two R-LPS vaccine candidates (Rev1Δ wzm and 16MΔ wzm ) with high susceptibility to polymyxins ( 27 ) to be investigated for safety and efficacy in the natural host.…”