Question

Brucellosis, caused by gram-negative Brucella species, is a zoonotic disease with serious impact on the livestock industry. In animals, brucellosis can lead to abortions in females and sterility in males. In humans, brucellosis is rarely fatal, but it does cause systemic febrile (fever-causing) disease that can be debilitating. Brucella bacteria enter macrophages but are able to evade phagolysosomal fusion and so are able to survive intracellularly. Brucella, unlike other gramnegative bacteria, such as E. coli, has an unusual LPS that does not activate the innate immune responses. Comparison of the sequenced genomes of several strains that are nonpathogenic to humans (B. neotomae and B. ovis) to those that are pathogenic to humans (B. melitensis, B. abortus, and B. suis) has revealed that Brucella species are highly conserved overall (90% of the genes share 98 to 100% sequence identity at the nucleotide level, with a GC% of 57.3), except for certain distinct regions, called genomic islands (GIs). A group of researchers hypothesized that host specicity and virulence differences must stem from the limited genome diversity found in ve of these regions that are present in all pathogenic strains: GI-1 (8.1 kb; 9 open reading frames [ORFs]; GC% 53.2), GI-2 (15.1 kb; 20 ORFs; GC% 51.3), GI-3 (21 kb; 30 ORFs; GC% 52.3), GI-5 (44.1 kb; 42 ORFs; GC% 57.2), and GI-6 (7.5 kb; 10 ORFs; GC% 54.2). Comparing these GIs among the species to that of the human pathogen B. melitensis, the researchers found that GI-6 is absent in B. neotomae and GI-1, GI-2, and GI-5 are absent in B. ovis. Below is a schematic diagram of the genetic organization of the ORFs within GI-2. Based on sequence homology to other genes in the DNA database, the researchers were able to annotate some of the ORFs present. The arrows indicate the direction of transcription. OMP, outer membrane protein; Tnpase, transposase

41-bp direct repeat 41-bp direct repeat EXTES Secretion protein Glycosyl transferase Tnpase Outer membrane protein tRNA ribosyl transferase Integrase tRNA Mannosyl transferase Tnpase

The researchers suspect that GI-2 may contain genes that contribute to the unusual LPS found in Brucella species. What led them to suspect this? (Hint: how does E. coli LPS stimulate innate immunity?)

Answer 1

The polysaccharide moiety of lipopolysaccharide or LPS primary plays protective roles for bacteria such as prevention from complement attacks or camouflage with common host carbohydrate residues.

In E.coli , the lipid moiety, termed lipid A, is recognized by the Toll-like receptor 4 (TLR4)/MD-2 complex, which transduces signals for activation of host innate immunity.

In Brucella, CHO reporter cell lines transfected with CD14 and TLRs showed that B. abortus triggers both TLR2 and TLR4. In contrast, lipopolysaccharide (LPS) and lipid A derived from Brucella rough (R) and smooth (S) strains activate CHO cells only through TLR4. Although Brucella LPS binds to TLR4, it does not induce the production of cytokines and antimicrobial peptides. Brucella LPS is several hundred times less effective at inducing the innate immunity than E. coli LPS, and this is thought to be important for Brucella to evade immune detection and to form a chronic intracellular infection.