Characterization of Brucella abortus O-Polysaccharide and Core Lipopolysaccharide Mutants and Demonstration that a Complete Core Is Required for Rough Vaccines To Be Efficient against Brucella abortus and Brucella ovis in the Mouse Model

Abstract

Brucella abortus rough lipopolysaccharide (LPS) mutants were obtained by transposon insertion into two wbk genes (wbkA [putative glycosyltransferase; formerly rfbU] and per [perosamine synthetase]), into manB (pmm [phosphomannomutase; formerly rfbK]), and into an unassigned gene. Consistent with gene-predicted roles, electrophoretic analysis, 2-keto-3-manno-D-octulosonate measurements, and immunoblots with monoclonal antibodies to O-polysaccharide, outer and inner core epitopes showed no O-polysaccharide expression and no LPS core defects in the wbk mutants. The rough LPS of manB mutant lacked the outer core epitope and the gene was designated manB core to distinguish it from the wbk manB O-Ag . The fourth gene (provisionally designated wa**) coded for a putative glycosyltransferase involved in inner core synthesis, but the mutant kept the outer core epitope. Differences in phage and polymyxin sensitivity, exposure or expression of outer membrane protein, core and lipid A epitopes, and lipid A acylation demonstrated that small changes in LPS core caused significant differences in B. abortus outer membrane topology. In mice, the mutants showed different degrees of attenuation and induced antibodies to rough LPS and outer membrane proteins. Core-defective mutants and strain RB51 were ineffective vaccines against B. abortus in mice. The mutants per and wbkA induced protection but less than the standard smooth vaccine S19, and controls suggested that anti O-polysaccharide antibodies accounted largely for the difference. Whereas no core-defective mutant was effective against B. ovis, S19, RB51, and the wbkA and per mutants afforded similar levels of protection. These results suggest that rough Brucella vaccines should carry a complete core for maximal effectiveness.Brucellosis is a zoonotic disease that causes heavy economic losses and human suffering. Under most conditions, vaccination and serological identification and culling of infected animals are the only practical means to achieve its eradication, but the best vaccines available (Brucella abortus S19 for cattle and B. melitensis Rev1 for sheep and goats) may induce abortions when used in pregnant animals and are virulent for humans. Moreover, like field strains, they carry a cell surface smoothtype lipopolysaccharide (S-LPS) whose immunodominant section (the N-formylperosamine O-polysaccharide) induces an antibody response that may be difficult to distinguish from that resulting from a true infection (25,48). This complicates serodiagnosis because the tests currently used detect antibodies to the O-polysaccharide.To overcome these problems, several strategies are possible. The early observation that rough (R) B. abortus strains are attenuated and do not agglutinate with antibody elicited by S bacteria (63) soon led to the concept of Brucella R vaccines and, more than 50 years ago, the spontaneous R mutant B. abortus 45/20 was studied for this purpose. However, strain 45/20 was unstable, and its use was abandoned (1, 48). The same strategy was followed to devel...

Rough vaccines in animal brucellosis: Structural and genetic basis and present status

“…the one followed by inert particles or non-virulent Brucella) [99], other factors related to the interplay of R mutanthost cells must be important, an aspect that has not been investigated so far. R mutants have altered outer membrane topology and LPS acylation patterns [78] and both features could be relevant in this regard.…”

“…But for the absence of an O-PS linked to the remaining LPS molecule, it can be predicted that not all these mutants are equivalent and they can be hypothetically grouped as follows: (i), R mutants that have a complete lipid A-core plus a cytoplasmic O-PS, the incorporation of which to the LPS is blocked (at least the wzm/wzt and possibly the waaL mutants); (ii), R mutants that have a complete lipid A-core but no O-PS (mutants in wb** glycosyltransferases, in wecA, and in genes coding for enzymes necessary for the synthesis of some precursors, such as manB OAg , gmd and per) and (iii), R mutants that have progressive deficiencies in the core and that may or may not accumulate cytoplasmic O-PS (mutants in some wa** genes and in some precursor genes such as manB core ). Mutants of each of these three groups have in fact been described [50,51,76,78,126], and the question then arises as to what extent they are equivalent in attenuation and immunizing abilities. Degree of core deficiency from R1 (R-LPS with complete core) to R3 (R-LPS devoid of at least the outer core sugars) as determined by SDS-PAGE and western blots with monoclonal antibodies to inner and outer core epitopes.…”

“…The B. abortus 45/20 and RB51 strains were developed in this way [75,112]. Alternatively, R mutants can be generated by Molecular Genetics methods such as transposon mutagenesis or deletion of genes involved in S-LPS biosynthesis [3,50,51,76,78,126,135].…”

Avaliação genética das vacinas contra a brucelose bovina comercializadas no Brasil

“…Brucella abortus é o agente etiológico da brucelose bovina causador de uma das doenças infecciosas que causam enormes perdas econômicas à cadeia da bovinocultura de corte e de leite, devido aos prejuízos causados em consequência dos distúrbios reprodutivos ocasionados nos animais (Poester et al 2002, Monreal et al 2003. Esta zoonose é responsável por problemas sanitários e econômicos, particularmente nos trópicos e em países com pouco investimento nas áreas de produção de leite e carne, onde a sua incidência é alta (Poester et al 2002).…”

Lipopolysaccharide as a target for brucellosis vaccine design

“…Also, mutants devoid of the O-PS (i.e. rough mutants) have been extensively investigated [18][19][20][21] (see rough vaccines below).…”