Shedding of Brucella melitensis happens through milk macrophages in the murine model of infection.

Although shedding of zoonotic brucellae in milk has been demonstrated in natural hosts, these data are still missing for the standard murine infection model. We therefore analysed shedding kinetics and the niche of B. melitensis in murine milk. Pregnant Balb/cByJ mice were intraperitoneally infected with 105 CFU of the 16 M reference strain, a 16 M mCherry mutant or a human isolate. Milk was collected over the course of lactation, and subjected to culture and immunofluorescence assays. Bacteria were also quantified in spleen and mammary glands of maternal mice and in spleen of the litter. The shedding of the three strains did not differ significantly (p = 0.301), ranging from log10 1.5 to 4.04 CFU/ml. A total of 73% of the mice excreted B. melitensis into the milk with peak values at mid-lactation; up to 30 bacteria/cell were found in macrophages and neutrophils. While the bacterial counts in the spleen of lactating females confirmed a well-established infection, only 50% of the pups harboured brucellae in their spleen, including the spleen of an uninfected pup fed by an infected foster mother. In conclusion, the murine model of infection may contribute to a better understanding of the zoonotic transmission of brucellosis.

HMGB1 release from trophoblasts contributes to inflammation during Brucella melitensis infection.

Brucella melitensis infection causes acute necrotizing inflammation in pregnant animals; however, the pathophysiological mechanisms leading to placentitis are unknown. Here, we demonstrate that high-mobility group box 1 (HMGB1) acts as a mediator of placenta inflammation in B. melitensis-infected pregnant mice model. HMGB1 levels were increased in trophoblasts or placental explant during B. melitensis infection. Inhibition of HMGB1 activity with neutralising antibody significantly reduced the secretion of inflammatory cytokines in B. melitensis-infected trophoblasts or placenta, whereas administration of recombinant HMGB1 (rHMGB1) increased the inflammatory response. Mechanistically, this decreased inflammatory response results from inhibition of HMGB1 activity, which cause the suppression of both mitogen-activated protein kinases and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. Moreover, neutralising antibody to HMGB1 prevented B. melitensis infection-induced activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in trophoblasts. In contrast, in vitro stimulation of trophoblasts with rHMGB1 caused activation of NADPH oxidase and increased the production of ROS, which contributes to high bacterial burden within trophoblasts or placenta. In vivo, treatment with anti-HMGB1 antibody increases the number of Brucella survival within placenta in B. melitensis-infected pregnant mice but successfully reduced the severity of placentitis and abortion.

A small non-coding RNA facilitates Brucella melitensis intracellular survival by regulating the expression of virulence factor.

Brucella species are the causative agents of brucellosis, a worldwide zoonotic disease that affects a broad range of mammals and causes great economic losses. Small regulatory RNAs (sRNAs) are post-transcriptional regulatory molecules that participate in the stress adaptation and pathogenesis of Brucella. In this study, we characterized the role of a novel sRNA, BSR1141, in the intracellular survival and virulence of Brucella melitensis. The results show that BSR1141 was highly induced during host infections and under in vitro stress situations that simulated the conditions encountered within host phagocytes. In addition, a BSR1141 mutant showed reduced survival both under in vitro stress conditions and in mice, confirming the role of BSR1141 in Brucella intracellular survival. Bioinformatic and experimental approaches revealed that BSR1141 affects the expression of many target genes, including the Brucella virulence component virB2. These data indicate that BSR1141 could influence the expression of virB2, which is important for B. melitensis pathogenesis and intracellular survival. This work provides new insight into the mechanism of adaptation to environmental stress and into the pathogenesis of intracellular pathogens.

Alterations of microRNAs and their predicted targeting mRNAs expression in RAW264.7 macrophages infected with Omp25 mutant Brucella melitensis.

Brucellosis is a worldwide zoonosis caused by Brucella species and represents a serious threat to both human and animal health. Omp25 is an important immunogenic and protective antigen in Brucella species; however, the functional mechanism of Omp25 in macrophages has not yet been elucidated. Here, we constructed a Brucella melitensis omp25 deletion mutant (M5-90-Δ omp25) and performed microRNA (miRNA) profiling of infected RAW264.7 cells. Eight differentially expressed miRNAs ( mmu-miR-146a-5p, mmu-miR-155-5p, mmu-miR-3473a, mmu-miR-149-3p, mmu-miR-671-5p, mmu-miR-1224-5p, mmu-miR-1895, and mmu-miR-5126) were identified, with quantitative real-time PCR (qRT-PCR) analysis confirming the up-regulation of mmu-miR-146-a-5p and mmu-miR-155-5p and down-regulation of mmu-miR-149-3p and mmu-miR-5126. mRNA profiling of B. melitensis M5-90-Δo mp25-infected RAW264.7 cells identified 967 differentially expressed genes (DEGs) (fold change ≥ 2). Among these, we focused on genes that were predicted by TargetScan, miRanda, and PicTar to be the potential targets of the differentially expressed miRNAs. The results suggested that 17 separate genes are potentially targeted by mmu-miR-149-3p, with one of these genes, Tbr1, also targeted by mmu-miR-5126. qRT-PCR analysis confirmed the up-regulation of nine of the predicted target genes. Our findings provide important information about the functional molecules in host cells, including miRNA and their target genes, affected by Omp25 from Brucella. This information is particularly valuable for the prophylaxis and treatment of brucellosis.

Brucella Melitensis 16M Regulates the Effect of AIR Domain on Inflammatory Factors, Autophagy, and Apoptosis in Mouse Macrophage through the ROS Signaling Pathway.

Brucellosis is a highly contagious zoonosis caused by Brucella. Brucella can invade and persist inside host cells, which results in chronic infection. We constructed AIR interference and overexpression lentiviruses to acquire AIR interference, overexpression, and rescue stable expression cell lines. We also established a Brucella melitensis 16M-infected macrophage model, which was treated with either the vehicle control or NAC (ROS scavenger N-acetylcysteine (NAC) for 0, 3, 6, 12, and 24 h. Confocal laser microscopy, transmission electron microscopy, fluorescence quantitative PCR, flow cytometry, ELISA, and Western blot were used to detect inflammation, cell autophagy and apoptosis-related protein expression levels, ROS levels, and the distribution of mitochondria. It was found that after interference and overexpression of AIR, ROS release was significantly changed, and mitochondria became abnormally aggregated. B. melitensis 16M activated the NLRP3/AIM2 inflammatory complex, and induced RAW264.7 cells to secrete IL-1ß and IL-18 through the ROS pathway. B. melitensis 16M also altered autophagy-related gene expression, increased autophagy activity, and induced cell apoptosis through the ROS pathway. The results showed that after B. melitensis 16M infection, ROS induced apoptosis, inflammation, and autophagy while AIR inhibited autophagosome maturation and autophagy initiation. Autophagy negatively regulated the activation of inflammasomes and prevented inflammation from occurring. In addition, mitophagy could promote cell apoptosis.

Nasal vaccination stimulates CD8(+) T cells for potent protection against mucosal Brucella melitensis challenge.

Brucellosis remains a significant zoonotic threat worldwide. Humans and animals acquire infection via their oropharynx and upper respiratory tract following oral or aerosol exposure. After mucosal infection, brucellosis develops into a systemic disease. Mucosal vaccination could offer a viable alternative to conventional injection practices to deter disease. Using a nasal vaccination approach, the ΔznuA B. melitensis was found to confer potent protection against pulmonary Brucella challenge, and reduce colonization of spleens and lungs by more than 2500-fold, with >50% of vaccinated mice showing no detectable brucellae. Furthermore, 10-fold more brucellae-specific, interferon-γ (IFN-γ)-producing CD8(+) T cells than CD4(+) T cells were induced in the spleen and respiratory lymph nodes. Evaluation of pulmonary and splenic CD8(+) T cells from mice vaccinated with ΔznuA B. melitensis revealed that these expressed an activated effector memory (CD44(hi)CD62L(lo)CCR7(lo)) T cells producing elevated levels of IFN-γ, tumor necrosis factor-α, perforin and granzyme B. To assess the relative importance of these increased numbers of CD8(+) T cells, CD8(-/-) mice were challenged with virulent B. melitensis, and they showed markedly increased bacterial loads in organs in contrast to similarly challenged CD4(-/-) mice. Only ΔznuA B. melitensis- and Rev-1-vaccinated CD4(-/-) and wild-type mice, not CD8(-/-) mice, were completely protected against Brucella challenge. Determination of cytokines responsible for conferring protection showed the relative importance of IFN-γ, but not interleukin-17 (IL-17). Unlike wild-type (wt) mice, IL-17 was greatly induced in IFN-γ(-/-) mice, but IL-17 could not substitute for IFN-γ's protection, although an increase in brucellae dissemination was observed upon in vivo IL-17 neutralization. These results show that nasal ΔznuA B. melitensis vaccination represents an attractive means to stimulate systemic and mucosal immune protection via CD8(+) T-cell engagement.

The effects of RegM on stress responses in Brucella melitensis.

Brucella melitensis is a facultative intracellular pathogen. The regM gene encodes a sensory transduction protein kinase in B. melitensis 16M, and genes orthologous to regM have been found to exist in many bacterial species. However, little is known about the regulation function of this gene in Brucella. In order to characterize this gene, we constructed a marked deletion mutant of regM as well as its complemented strain. The mutant was less able to withstand acid and hyperosmotic conditions than wild-type strain but shown no significant difference with wild-type strain when challenged by elevated temperature and hypotonic conditions. In addition, inactivation of regM did not affect virulence in B. melitensis 16M in macrophage and mice infection models.

Quorum sensing and self-quorum quenching in the intracellular pathogen Brucellamelitensis.

Brucella quorum sensing has been described as an important regulatory system controlling crucial virulence determinants such as the VirB type IV secretion system and the flagellar genes. However, the basis of quorum sensing, namely the production of autoinducers in Brucella has been questioned. Here, we report data obtained from the use of a genetic tool allowing the in situ detection of long-chain N-acyl-homoserine lactones (AHL) activity at single bacterium level in Brucella melitensis. These data are consistent with an intrinsic production of AHL by B. melitensis in low concentration both during in vitro growth and macrophage infection. Moreover, we identified a protein, named AibP, which is homologous to the AHL-acylases of various bacterial species. In vitro and during infection, expression of aibP coincided with a decrease in endogenous AHL activity within B. melitensis, suggesting that AibP could efficiently impair AHL accumulation. Furthermore, we showed that deletion of aibP in B. melitensis resulted in enhanced virB genes expression and VirB8 production as well as in a reduced flagellar genes expression and production of FlgE (hook protein) and FliC (flagellin) in vitro. Altogether, these results suggest that AHL-dependent quorum sensing and AHL-quorum quenching coexist in Brucella, at least to regulate its virulence.

Brucella induces an unfolded protein response via TcpB that supports intracellular replication in macrophages.

Brucella melitensis is a facultative intracellular bacterium that causes brucellosis, the most prevalent zoonosis worldwide. The Brucella intracellular replicative niche in macrophages and dendritic cells thwarts immune surveillance and complicates both therapy and vaccine development. Currently, host-pathogen interactions supporting Brucella replication are poorly understood. Brucella fuses with the endoplasmic reticulum (ER) to replicate, resulting in dramatic restructuring of the ER. This ER disruption raises the possibility that Brucella provokes an ER stress response called the Unfolded Protein Response (UPR). In this study, B. melitensis infection up regulated expression of the UPR target genes BiP, CHOP, and ERdj4, and induced XBP1 mRNA splicing in murine macrophages. These data implicate activation of all 3 major signaling pathways of the UPR. Consistent with previous reports, XBP1 mRNA splicing was largely MyD88-dependent. However, up regulation of CHOP, and ERdj4 was completely MyD88 independent. Heat killed Brucella stimulated significantly less BiP, CHOP, and ERdj4 expression, but induced XBP1 splicing. Although a Brucella VirB mutant showed relatively intact UPR induction, a TcpB mutant had significantly compromised BiP, CHOP and ERdj4 expression. Purified TcpB, a protein recently identified to modulate microtubules in a manner similar to paclitaxel, also induced UPR target gene expression and resulted in dramatic restructuring of the ER. In contrast, infection with the TcpB mutant resulted in much less ER structural disruption. Finally, tauroursodeoxycholic acid, a pharmacologic chaperone that ameliorates the UPR, significantly impaired Brucella replication in macrophages. Together, these results suggest Brucella induces a UPR, via TcpB and potentially other factors, that enables its intracellular replication. Thus, the UPR may provide a novel therapeutic target for the treatment of brucellosis. These results also have implications for other intracellular bacteria that rely on host physiologic stress responses for replication.

The effects of MucR on expression of type IV secretion system, quorum sensing system and stress responses in Brucella melitensis.

MucR is a transcriptional regulator in many bacterial pathogens and is required for virulence in mice and macrophages, resistance to stress responses, and modification of the cell envelope in Brucella spp. To determine why the mucR deleted mutant is attenuated in vivo and in vitro, we performed RNA-seq analysis using Brucella melitensis RNA obtained from B. melitensis 16M and 16MΔmucR grown under the same conditions. We found 442 differentially expressed genes; 310 were over expressed, and 132 were less expressed in 16MΔmucR. Many genes identified are involved in metabolism, cell wall/envelope biogenesis, replication, and translation. Notably, genes involved in type IV secretion system and quorum sensing system were down-regulated in 16MΔmucR. In addition, genes involved in tolerance to acid and iron-limitation were also affected and experimentally verified in this study. The effects of MucR on Brucella survival and persistence in mice and macrophages were related to type IV secretion system, quorum sensing system, and stress tolerance, which also provide added insight to the MucR regulon.

Impact of Hfq on global gene expression and intracellular survival in Brucella melitensis.

Brucella melitensis is a facultative intracellular bacterium that replicates within macrophages. The ability of brucellae to survive and multiply in the hostile environment of host macrophages is essential to its virulence. The RNA-binding protein Hfq is a global regulator that is involved in stress resistance and pathogenicity. Here we demonstrate that Hfq is essential for stress adaptation and intracellular survival in B. melitensis. A B. melitensis hfq deletion mutant exhibits reduced survival under environmental stresses and is attenuated in cultured macrophages and mice. Microarray-based transcriptome analyses revealed that 359 genes involved in numerous cellular processes were dysregulated in the hfq mutant. From these same samples the proteins were also prepared for proteomic analysis to directly identify Hfq-regulated proteins. Fifty-five proteins with significantly affected expression were identified in the hfq mutant. Our results demonstrate that Hfq regulates many genes and/or proteins involved in metabolism, virulence, and stress responses, including those potentially involved in the adaptation of Brucella to the oxidative, acid, heat stress, and antibacterial peptides encountered within the host. The dysregulation of such genes and/or proteins could contribute to the attenuated hfq mutant phenotype. These findings highlight the involvement of Hfq as a key regulator of Brucella gene expression and facilitate our understanding of the role of Hfq in environmental stress adaptation and intracellular survival of B. melitensis.

Osteoarticular tissue infection and development of skeletal pathology in murine brucellosis.

Brucellosis, a frequent bacterial zoonosis, can produce debilitating chronic disease with involvement of multiple organs in human patients. Whereas acute brucellosis is well studied using the murine animal model, long-term complications of host-pathogen interaction remain largely elusive. Human brucellosis frequently results in persistent, chronic osteoarticular system involvement, with complications such as arthritis, spondylitis and sacroiliitis. Here, we focused on identifying infectious sites in the mouse that parallel Brucella melitensis foci observed in patients. In vivo imaging showed rapid bacterial dispersal to multiple sites of the murine axial skeleton. In agreement with these findings, immunohistochemistry revealed the presence of bacteria in bones and limbs, and in the lower spine vertebrae of the axial skeleton where they were preferentially located in the bone marrow. Surprisingly, some animals developed arthritis in paws and spine after infection, but without obvious bacteria in these sites. The identification of Brucella in the bones of mice corroborates the findings in humans that these osteoarticular sites are important niches for the persistence of Brucella in the host, but the mechanisms that mediate pathological manifestations in these sites remain unclear. Future studies addressing the immune responses within osteoarticular tissue foci could elucidate important tissue injury mediators and Brucella survival strategies.

The two-component system PrlS/PrlR of Brucella melitensis is required for persistence in mice and appears to respond to ionic strength.

Bacterial adaptation to environmental conditions is essential to ensure maximal fitness in the face of several stresses. In this context, two-component systems (TCSs) represent a predominant signal transduction mechanism, allowing an appropriate response to be mounted when a stimulus is sensed. As facultative intracellular pathogens, Brucella spp. face various environmental conditions, and an adequate response is required for a successful infection process. Recently, bioinformatic analysis of Brucella genomes predicted a set of 15 bona fide TCS pairs, among which some have been previously investigated. In this report, we characterized a new TCS locus called prlS/R, for probable proline sensor-regulator. It encodes a hybrid histidine kinase (PrlS) with an unusual Na(+)/solute symporter N-terminal domain and a transcriptional regulator (belonging to the LuxR family) (PrlR). In vitro, Brucella spp. with a functional PrlR/S system form bacterial aggregates, which seems to be an adaptive response to a hypersaline environment, while a prlS/R mutant does not. We identified ionic strength as a possible signal sensed by this TCS. Finally, this work correlates the absence of a functional PrlR/S system with the lack of hypersaline-induced aggregation in particular marine Brucella spp.

MicroRNA expression profile in RAW264.7 cells in response to Brucella melitensis infection.

MicroRNA (miRNA) is small non-coding RNA with approximate 22 nt in length. Recent studies indicate that miRNAs play significant roles in pathogen-host interactions. Brucella organisms are Gram-negative facultative intracellular bacteria that cause Brucellosis. Brucella strains infect macrophages and establish chronic infection by altering host life activities including apoptosis and autophagy. Here, we report a comprehensive analysis of miRNA expression profiles in mock- and Brucella-infected RAW264.7 cells using high-throughput sequencing approach. In total, 344 unique miRNAs were co-expressed in the two libraries, in which 57 miRNAs were differentially expressed. Eight differentially expressed miRNAs with high abundance were subjected to further analysis. The GO enrichment analysis suggests that the putative target genes of these differentially expressed miRNAs are involved in apoptosis, autophagy and immune response. In particular, a total of 25 target genes are involved in regulating apoptosis and autophagy, indicating that these miRNAs may play important regulatory roles in the Brucella-host interactions. Furthermore, the interactions of miR-1981 and its target genes, Bcl-2 and Bid, were validated by luciferase assay. The results show that miR-1981 mimic up-regulated the luciferase activity of psiCHECK-2 Bcl-2 3' UTR, but the luciferase activity of psiCHECK-2 Bid 3' UTR was not changed significantly. Taken together, these data provide valuable framework on Brucella induced miRNA expression in RAW264.7 cells, and suggest that Brucella may establish chronic infection by regulating miRNA expression profile.

Differential expression of iron acquisition genes by Brucella melitensis and Brucella canis during macrophage infection.

Brucella spp. cause chronic zoonotic disease often affecting individuals and animals in impoverished economic or public health conditions; however, these bacteria do not have obvious virulence factors. Restriction of iron availability to pathogens is an effective strategy of host defense. For brucellae, virulence depends on the ability to survive and replicate within the host cell where iron is an essential nutrient for the growth and survival of both mammalian and bacterial cells. Iron is a particularly scarce nutrient for bacteria with an intracellular lifestyle. Brucella melitensis and Brucella canis share ~99% of their genomes but differ in intracellular lifestyles. To identify differences, gene transcription of these two pathogens was examined during infection of murine macrophages and compared to broth grown bacteria. Transcriptome analysis of B. melitensis and B. canis revealed differences of genes involved in iron transport. Gene transcription of the TonB, enterobactin, and ferric anguibactin transport systems was increased in B. canis but not B. melitensis during infection of macrophages. The data suggest differences in iron requirements that may contribute to differences observed in the lifestyles of these closely related pathogens. The initial importance of iron for B. canis but not for B. melitensis helps elucidate differing intracellular survival strategies for two closely related bacteria and provides insight for controlling these pathogens.

Deep sequencing-based expression transcriptional profiling changes during Brucella infection.

Brucellosis is a worldwide zoonotic infectious disease that has significant economic effects on animal production and human health. The host macrophage -Brucella interaction is critical to the establishment of infections. Thus, the kinetic transcriptional profile of gene expression in macrophages infected with the Brucella melitensis strain 16M was investigated in the current study using a technology based on deep sequencing. The total RNA was extracted from macrophages 0, 4, and 24 h post-infection. Data analysis showed that in the gene ontology term, the expression of genes in the endoplasmic reticulum, lysosomes, as well as those involved in programmed cell death and apoptosis significantly changed during the first 24 h post-infection. Pathway enrichment analysis indicated that the genes in the apoptosis pathway, NOD-like receptor signaling pathway, Fc gamma R-mediated phagocytosis, lysosome pathway, p53 signaling pathway, and protein processing in the endoplasmic reticulum significantly changed during the first 24 h post-infection. The B-cell receptor and toll-like receptor signaling pathways were also significantly changed 24 h post-infection compared with those 4 h post-infection. The results of the current study can contribute to an improved understanding of the manner by which host cell responses may be manipulated to prevent Brucella infection.

Nondividing but metabolically active gamma-irradiated Brucella melitensis is protective against virulent B. melitensis challenge in mice.

Brucella spp. are gram-negative bacteria that cause the most frequent zoonotic disease worldwide, with more than 500,000 human infections yearly; however, no human vaccine is currently available. As with other intracellular organisms, cytotoxic mechanisms against infected cells are thought to have an important role in controlling infection and mediating long-term immunity. Live attenuated strains developed for use in animals elicit protection but retain unacceptable levels of virulence. Thus, the optimal design for a brucellosis vaccine requires a nonliving vaccine that confers effective immunity. Historically, inactivation methods such as chemical or heat treatment successfully impair Brucella reproductive capacity; nevertheless, metabolically inactive vaccines (subunit or killed) present very limited efficacy. Hence, we hypothesized that bacterial metabolism plays a major role in creating the proper antigenic and adjuvant properties required for efficient triggering of protective responses. Here, we demonstrate that inactivation of Brucella melitensis by gamma-irradiation inhibited its replication capability and yet retained live-Brucella protective features. Irradiated Brucella possessed metabolic and transcriptional activity, persisted in macrophages, generated antigen-specific cytotoxic T cells, and protected mice against virulent bacterial challenge, without signs of residual virulence. In conclusion, pathogen metabolic activity has a positive role in shaping protective responses, and the generation of inactivated and yet metabolically active microbes is a promising strategy for safely vaccinating against intracellular organisms such as B. melitensis.

Persistence of an atherogenic lipid profile after treatment of acute infection with Brucella.

Serum lipid changes during infection may be associated with atherogenesis. No data are available on the effect of Brucellosis on lipids. Lipid parameters were determined in 28 patients with Brucellosis on admission and 4 months following treatment and were compared with 24 matched controls. Fasting levels of total cholesterol (TC), HDL-cholesterol (HDL-C), triglycerides, apolipoproteins (Apo) A, B, E CII, and CIII, and oxidized LDL (oxLDL) were measured. Activities of serum cholesterol ester transfer protein (CETP), paraoxonase 1 (PON1), and lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) and levels of cytokines [interleukins (IL)-1beta, IL-6, and tumor necrosis factor (TNFa)] were also determined. On admission, patients compared with controls had 1) lower levels of TC, HDL-C, LDL-cholesterol (LDL-C), ApoB, ApoAI, and ApoCIII and higher LDL-C/HDL-C and ApoB/ApoAI ratios; 2) higher levels of IL-1b, IL-6, and TNFa; 3) similar ApoCII and oxLDL levels and Lp-PLA(2) activity, lower PON1, and higher CETP activity; and 4) higher small dense LDL-C concentration. Four months later, increases in TC, HDL-C, LDL-C, ApoB, ApoAI, and ApoCIII levels, ApoB/ApoAI ratio, and PON1 activity were noticed compared with baseline, whereas CETP activity decreased. LDL-C/HDL-C ratio, ApoCII, and oxLDL levels, Lp-PLA(2) activity, and small dense LDL-C concentration were not altered. Brucella infection is associated with an atherogenic lipid profile that is not fully restored 4 months following treatment.

Genomic island 2 of Brucella melitensis is a major virulence determinant: functional analyses of genomic islands.

Brucella genomic islands (GIs) share similarities in their genomic organization to pathogenicity islands from other bacteria and are likely acquired by lateral gene transfer. Here, we report the identification of a GI that is important for the pathogenicity of Brucella melitensis. The deletion of GI-1, GI-5, or GI-6 did not affect bacterial growth in macrophages as well as their virulence in interferon regulatory factor 1-deficient (IRF-1(-/-)) mice, suggesting that these islands do not contribute to Brucella virulence. However, the deletion of GI-2 resulted in the attenuation of bacterial growth in macrophages and virulence in IRF-1(-/-) mice. The GI-2 mutant also displayed a rough lipopolysaccharide (LPS) phenotype indicated by acriflavin agglutination, suggesting that in vitro and in vivo attenuation is a result of LPS alteration. Further, systematic analysis of the entire GI-2 revealed two open reading frames (ORFs), BMEI0997 and I0998, that encode hypothetical sugar transferases and contribute to LPS alteration, as the deletion of either of these ORFs resulted in a rough phenotype similar to that of the GI-2 mutant. Complementation analyses indicated that in addition to I0997 and I0998, I0999 is required to restore the smooth LPS in the GI-2 mutant as well as its full in vitro and in vivo virulence. The I0999 sequence analysis suggested that it might function as a transporter to help facilitate the transport or linking of the O antigen to the LPS. Our study also indicated that the rough LPS resulting from the GI-2 deletion may affect pathogen-associated molecular pattern recognition by Toll-like receptors.