Cross-sectional associations of low body mass index and being underweight with joint damage in Chinese patients with rheumatoid arthritis.

Objective: To characterize BMI in Chinese patients with RA vs US patients and examine its association with joint damage in Chinese patients. Methods: Each of the 1318 patients from a real-world Chinese RA population was first stratified by gender and then individually age-matched with one American RA patient from the US National Health and Nutritional Examination Survey 1999-2018. Data on BMI, bilateral hand radiographs and risk factors at enrolment were collected but radiographs were unavailable for the American patients. Logistic regression was used to evaluate the association of BMI with radiographic joint damage (RJD) in Chinese patients. Results: Chinese patients had a significantly lower BMI [(weighted) median 21.8 vs 29.8 kg/m2; P < 0.001] and a higher prevalence of being underweight (15.2% vs 1.1%; P < 0.05) than their American counterparts. Underweight Chinese patients (BMI <18.5) had higher modified total Sharp scores (median 17 vs 10) and joint space narrowing (JSN) subscores (median 6 vs 2) (both P < 0.05) than normal-weight patients (BMI ≥18.5-<24). After controlling for confounding, continuous BMI was cross-sectionally negatively associated with RJD [adjusted prevalence odds ratio (OR) 0.90 (95% CI 0.85, 0.96)] and JSN [adjusted prevalence OR 0.92 (95% CI 0.87, 0.96)]; being underweight vs normal weight was associated with RJD [adjusted prevalence OR 2.14 (95% CI 1.37, 3.35)] and JSN [adjusted prevalence OR 1.77 (95% CI 1.10, 2.84)]. Conclusion: Low BMI and being underweight were cross-sectionally associated with joint damage in Chinese RA patients, especially JSN, suggesting the clinical importance of identifying underweight patients and focusing on weight gain to prevent joint damage.

Assessment of the hypothetical protein BB0616 in the murine infection of Borrelia burgdorferi.

bb0616 of Borrelia burgdorferi, the Lyme disease pathogen, encodes a hypothetical protein of unknown function. In this study, we showed that BB0616 was not surface-exposed or associated with the membrane through localization analyses using proteinase K digestion and cell partitioning assays. The expression of bb0616 was influenced by a reduced pH but not by growth phases, elevated temperatures, or carbon sources during in vitro cultivation. A transcriptional start site for bb0616 was identified by using 5' rapid amplification of cDNA ends, which led to the identification of a functional promoter in the 5' regulatory region upstream of bb0616. By analyzing a bb0616-deficient mutant and its isogenic complemented counterparts, we found that the infectivity potential of the mutant was significantly attenuated. The inactivation of bb0616 displayed no effect on borrelial growth in the medium or resistance to oxidative stress, but the mutant was significantly more susceptible to osmotic stress. In addition, the production of global virulence regulators such as BosR and RpoS as well as virulence-associated outer surface lipoproteins OspC and DbpA was reduced in the mutant. These phenotypes were fully restored when gene mutation was complemented with a wild-type copy of bb0616. Based on these findings, we concluded that the hypothetical protein BB0616 is required for the optimal infectivity of B. burgdorferi, potentially by impacting B. burgdorferi virulence gene expression as well as survival of the spirochete under stressful conditions.

Geriatric nutrition risk index: a more powerful index identifying muscle mass loss in patients with rheumatoid arthritis.

OBJECTIVE: To explore the association of geriatric nutrition risk index (GNRI), a traditional albumin-body weight calculation, with myopenia in patients with rheumatoid arthritis (RA) and compare its ability to identify myopenia with protein indicators. METHODS: This cross-sectional study was carried out based on a Chinese RA cohort. Clinical data and protein indicators (including albumin, globulin, albumin to globulin ratio, prealbumin, hemoglobin) were collected. GNRI was estimated by serum albumin and body weight. Myopenia was indicated as muscle mass loss measured by bioelectric impedance analysis. RESULTS: There were 789 RA patients included with mean age 52.6 ± 12.6 years and 77.6% female. There were 41.3%, 18.0%, 27.5%, 13.2% patients with no (GNRI > 98), low (GNRI 92 to ≤ 98), moderate (GNRI 82 to < 92), and major nutrition-related risk (GNRI < 82). There were 406 (51.5%) RA patients with myopenia, RA patients with major nutrition-related risk had the highest prevalence of myopenia (87.5% vs. 73.3% vs. 50.0% vs. 26.1%). Multivariate logistic analysis showed that compared with no risk, RA patients with low (OR = 3.23, 95% CI: 1.86-5.61), moderate (OR = 9.56, 95% CI: 5.70-16.01), and major nutrition-related risk (OR = 28.91, 95% CI: 13.54-61.71) were associated with higher prevalence of myopenia. Receiver operating characteristic curves showed that GNRI (AUC = 0.79) performed a better identifiable ability toward myopenia than serum albumin (AUC = 0.66) or others indicators (AUC range 0.59 to 0.65), respectively. CONCLUSION: GNRI, an objective and convenient albumin-weight index, may be preferable for identifying myopenia in RA patients. Key Points • We firstly elucidated the association of GNRI with muscle mass loss among RA patients, and compared its ability to identify muscle mass loss with serum albumin or other protein indicators. • Major nutrition-related risk identified by GNRI showed the highest risk of muscle mass loss, GNRI demonstrated a greater ability to identify myopenia in RA patients. which indicated GNRI was an objective and convenient albumin-weight index to identify myopenia in RA patients.

Role of the Hypothetical Protein BB0563 during Borrelia burgdorferi Infection in Animals.

The alternative sigma factor RpoS in Borrelia burgdorferi, the etiological agent of Lyme disease, has long been postulated to regulate virulence-associated genes other than ospC and dbpA. Here, we demonstrate that bb0563, a gene encoding a hypothetical protein, is regulated by RpoS and contributes to the optimal infectivity of B. burgdorferi. When B. burgdorferi was exposed to environmental stimuli, bb0563 showed similar expression patterns as rpoS, ospC, and dbpA. Expression of bb0563 was significantly downregulated when rpoS was inactivated and was restored in the complemented strain. By using rapid amplification of cDNA ends (RACE) and luciferase reporter assays, a functional promoter was identified in the regulatory region upstream of bb0563. Gene expression from this promoter was drastically decreased in the rpoS mutant. We next investigated the role of bb0563 during animal infection. By using quantitative reverse transcription-PCR (RT-PCR), we found that bb0563 was highly expressed in mouse tissues during infection. We further created a bb0563-deficient mutant in a bioluminescent B. burgdorferi strain and examined infection dynamics using in vivo imaging. Relative to the parental and complemented strains, the mutant showed a delayed infection pattern and bacterial load was reduced. Another bb0563 deletion mutant was also created in the strain 297 background, and quantitative PCR (qPCR) analysis revealed a significantly lower spirochetal burden in tissue samples collected from animals infected with the mutant. In addition, localization studies indicate that BB0563 is not exposed on the cell surface but is associated with outer membrane. Taken together, these results suggest that bb0563 is required for optimal infectivity of B. burgdorferi during experimental infection.

[Clinical characteristics and genetic analysis of a Chinese pedigree affected by glycogen storage disease type Ia with gout as the first manifestation].

OBJECTIVE: To explore the clinical and genetic characteristics of a Chinese pedigree affected by glycogen storage disease (GSD) type Ia with gout as the first manifestation. METHODS: Clinical and biochemical data of the pedigree were collected. Available members of the pedigree were subjected to gene sequencing, and the result was analyzed by bioinformatics software. The pedigree was followed up for five years. RESULTS: The proband was a young female manifesting recurrent gout flare, hypoglycemia, and hypertriglyceridemia. One of her younger brothers also presented with dysplasia and hepatic adenoma. Gene sequencing revealed that the proband and her younger brother both harbored c.1022T>A (p.I1e341Asn) and c.230+5G>A compound heterozygous variants of the G6PC gene , which were inherited from their father and mother, respectively. Among these, the c.230+5G>A is an intron region variant which was unreported previously, and bioinformatics analysis showed that it may impact mRNA splicing of the gene. The proband was treated with raw corn starch, allopurinol, and fenofibrate. Gout was well controlled, and she had given birth to a baby girl without GSD. CONCLUSION: GSD Ia should be considered among young gout patients with hypoglycemia and hepatomegaly, for which gene sequencing is warranted. GSD Ia has a good prognosis after comprehensive treatment with diet and medicine.

BB0761, a MepM homolog, contributes to Borrelia burgdorferi cell division and mammalian infectivity.

M23 family endopeptidases play important roles in cell division and separation in a wide variety of bacteria. Recent studies have suggested that these proteins also contribute to bacterial virulence. However, the biological function of M23 peptidases in pathogenic spirochetes remains unexplored. Here, we describe Borrelia burgdorferi, the bacterial pathogen causing Lyme disease, requires a putative M23 family homolog, BB0761, for spirochete morphology and cell division. Indeed, the inactivation of bb0761 led to an aberrant filamentous phenotype as well as the impairment of B. burgdorferi growth in vitro. These phenotypes were complemented not only with B. burgdorferi bb0761, but also with the mepM gene from E. coli. Moreover, the bb0761 mutant showed a complete loss of infectivity in a murine model of Lyme borreliosis. Resistance of the mutant to osmotic and oxidative stresses was markedly reduced. Our combined results indicate that BB0761 contributes to B. burgdorferi cell division and virulence.

A Matrix Prediction Model for the 6-Month Mortality Risk in Patients With Anti-Melanoma Differentiation-Associated Protein-5-Positive Dermatomyositis.

Objectives: The purpose of this study was to investigate the baseline independent risk factors for predicting 6-month mortality of patients with anti-melanoma differentiation-associated gene 5 (anti-MDA5)-positive dermatomyositis (DM) and develop a matrix prediction model formed by these risk factors. Methods: The hospitalized patients with DM who completed at least 6-month follow-up were recruited as a derivation cohort. The primary exposure was defined as positive anti-MDA5 at the baseline. The primary outcome was all-cause 6-month mortality after enrollment. A matrix prediction model was developed in the derivation cohort, and another published cohort was used for external validation. Results: In derivation cohort, 82 patients with DM were enrolled (mean age of onset 50 ± 11 years and 63% women), with 40 (49%) showing positive anti-MDA5. Gottron sign/papules (OR: 5.135, 95%CI: 1.489-17.708), arthritis (OR: 5.184, 95%CI: 1.455-18.467), interstitial lung disease (OR: 7.034, 95%CI: 1.157-42.785), and higher level of C4 (OR: 1.010, 95%CI: 1.002-1.017) were the independent associators with positive anti-MDA5 in patients with DM. Patients with anti-MDA5-positive DM had significant higher 6-month all-cause mortality than those with anti-MDA5-negative (30 vs. 0%). Among the patients with anti-MDA5-positive DM, compared to the survivors, non-survivors had significantly advanced age of onset (59 ± 6 years vs. 46 ± 9 years), higher rates of fever (75 vs. 18%), positive carcinoma embryonic antigen (CEA, 75 vs. 14%), higher level of ferritin (median 2,858 ug/L vs. 619 ug/L, all p < 0.05). A stepwise multivariate Cox regression showed that ferritin ≥1,250 µg/L (HR: 10.4, 95%CI: 1.8-59.9), fever (HR: 11.2, 95%CI: 2.5-49.9), and positive CEA (HR: 5.2, 95%CI: 1.0-25.7) were the independent risk factors of 6-month mortality. A matrix prediction model was built to stratify patients with anti-MDA5-positive DM into different subgroups with various probabilities of 6-month mortality risk. In an external validation cohort, the observed 6-month all-cause mortality was 78% in high-risk group, 43% in moderate-risk group, and 25% in low-risk group, which shows good accuracy of the model. Conclusion: Baseline characteristics such as fever, ferritin ≥1,250 µg/L, and positive CEA are the independent risk factors for 6-month all-cause mortality in patients with anti-MDA5-positive DM. A novel matrix prediction model composed of these three clinical indicators is first proposed to provide a chance for the exploration of individual treatment strategies in anti-MDA5-positive DM subgroups with various probabilities of mortality risk.

Gene Regulation and Transcriptomics.

Borrelia (Borreliella) burgdorferi, along with closely related species, is the etiologic agent of Lyme disease. The spirochete subsists in an enzootic cycle that encompasses acquisition from a vertebrate host to a tick vector and transmission from a tick vector to a vertebrate host. To adapt to its environment and persist in each phase of its enzootic cycle, B. burgdorferi wields three systems to regulate the expression of genes: the RpoN-RpoS alternative sigma factor cascade, the Hk1/Rrp1 two-component system and its product c-di-GMP, and the stringent response mediated by RelBbu and DksA. These regulatory systems respond to enzootic phase-specific signals and are controlled or fine- tuned by transcription factors, including BosR and BadR, as well as small RNAs, including DsrABb and Bb6S RNA. In addition, several other DNA-binding and RNA-binding proteins have been identified, although their functions have not all been defined. Global changes in gene expression revealed by high-throughput transcriptomic studies have elucidated various regulons, albeit technical obstacles have mostly limited this experimental approach to cultivated spirochetes. Regardless, we know that the spirochete, which carries a relatively small genome, regulates the expression of a considerable number of genes required for the transitions between the tick vector and the vertebrate host as well as the adaptation to each.

DksA plays an essential role in regulating the virulence of Borrelia burgdorferi.

The RNA polymerase-binding protein DksA, together with the alarmone nucleotides (p)ppGpp, mediates the stringent response to nutrient starvation in Borrelia burgdorferi. To date, the contribution of DksA to B. burgdorferi infection remains unknown. We report here that DksA is essential for B. burgdorferi to infect a mammalian host. dksA expression was highly induced during infection. Moreover, a dksA-deficient mutant was incapable of infecting mice. The mutant displayed growth defects when cultured in vitro and resistance to osmotic pressure was markedly reduced. These phenotypes were fully restored to those of the wild type when dksA mutation was complemented. We further showed that DksA controlled the expression of virulence-associated lipoprotein OspC, likely via the central alternative sigma factor RpoS. Synthesis of RpoS was abolished in the dksA mutant, but rpoS transcription remained unaffected. Additionally, we found that the expression of clpX, clpA, clpP, and clpP2 was significantly increased in the mutant, suggesting that DksA may post-transcriptionally regulate rpoS expression via its effect on ClpXP and/or ClpAP proteases. These combined data demonstrate that DksA regulates B. burgdorferi virulence at least partially through its influence on RpoS and OspC. This study thus elucidates that, in addition to function as a stringent response regulator, DksA promotes the transcription and/or translation of genes contributing to B. burgdorferi infectivity.

The Lon-1 Protease Is Required by Borrelia burgdorferi To Infect the Mammalian Host.

Borrelia burgdorferi encodes a functional homolog of canonical Lon protease termed Lon-2. In addition, B. burgdorferi encodes a second Lon homolog called Lon-1. Recent studies suggest that Lon-1 may function differently from the prototypical Lon protease. However, the function of Lon-1 in B. burgdorferi biology remains virtually unknown. Particularly, the contribution of Lon-1 to B. burgdorferi fitness and infection remains hitherto unexplored. Herein, we show that Lon-1 plays a critical role for the infection of B. burgdorferi in a mammalian host. We found that lon-1 was highly expressed during animal infection, implying an important function of this protein in bacterial infection. We further generated a lon-1 deletion mutant and an isogenic complemented strain. Relative to that of the wild-type strain, the infectivity of the mutant was severely attenuated in a murine infection model. Our data also showed that the mutant displayed growth defects in regular BSK-II medium. Furthermore, bacterial resistance to osmotic stress was markedly reduced when lon-1 was inactivated. When exposed to tert-butyl hydroperoxide, survival of the lon-1 mutant was impaired. In addition, production of several virulence factors, such as BosR, RpoS, and OspC, was elevated in the mutant. These phenotypes were restored when the lon-1 mutation was complemented. Finally, we created a lon-1(S714A) mutant and found that this mutant failed to infect mice, suggesting that the proteolytic activity of Lon-1 is essential for bacterial infection. Taken together, these results demonstrate that Lon-1 is required by B. burgdorferi to infect animal hosts and to cope with environmental stresses.

The Lon-2 protease of Borrelia burgdorferi is critical for infection in the mammalian host.

Borrelia burgdorferi encodes a functional homolog of canonical Lon protease termed Lon-2. To date, the contribution of Lon-2 to B. burgdorferi fitness and infection remains unexplored. Herein, we showed that expression of lon-2 was highly induced during animal infection, suggesting that Lon-2 is important for B. burgdorferi infection. We further generated a lon-2 deletion mutant. Compared with that of wild-type (WT) strain, the infectivity of the mutant was severely attenuated in a murine infection model. Although no growth defect was observed for the mutant in normal BSK-II medium, resistance of the lon-2 mutant to osmotic stress was markedly reduced. In addition, when exposed to tert-Butyl hydroperoxide, survival of the lon-2 mutant was impaired. In addition, we found that the protein levels of RpoS and RpoS-dependent OspC were decreased in the mutant. All these phenotypes were restored to WT or near-WT levels when lon-2 mutation was complemented in cis. Taken together, these results demonstrate that Lon-2 is critical for B. burgdorferi to establish infection and to cope with environmental stresses. This study provides a foundation for further uncovering the direct link between the dual roles of Lon-2 in protein quality control and bacterial pathogenesis.

The CXXC Motifs Are Essential for the Function of BosR in Borrelia burgdorferi.

BosR, a Fur family member, is essential for the pathogenesis of the Lyme disease pathogen, Borrelia burgdorferi. Unlike typical Fur proteins in which DNA binding represses gene expression, binding of BosR to the rpoS promoter directly activates rpoS transcription in B. burgdorferi. However, virtually nothing is known concerning potential structural features and amino acid residues of BosR that are important for protein function and virulence regulation in B. burgdorferi. Particularly, it remains unknown what structural motifs or residues of BosR coordinate Zn, although previous analyses have indicated that the function of BosR may depend on Zn. To address these information gaps, we herein introduced mutations into four conserved cysteine residues in two putative CXXC motifs of BosR. Our data showed that the ability of BosR to bind Zn was dramatically reduced when the CXXC motifs were mutated. Moreover, we found that the two CXXC motifs contributed to the ability of BosR to form dimers. By using a trans-complementation genetic approach, we additionally demonstrated that both CXXC motifs of BosR were essential for in vivo gene expression regulation. Mutation of any of the four cysteines abolished the transcriptional activation of rpoS. In contrast to wild type BosR, each mutant protein was incapable of binding the rpoS promoter in electrophoretic mobility shift assays. The combined data strongly support that the two CXXC motifs and four cysteines constitute the structural site essential for Zn-coordination, protein dimerization, and the unique regulatory activity of BosR.

MyD88 Signaling in T Cells Is Critical for Effector CD4 T Cell Differentiation following a Transitional T Follicular Helper Cell Stage.

Activation of CD4 T cells by dendritic cells leads to their differentiation into various effector lineages. The nature of the effector lineage is determined by the innate cues provided by dendritic cells to newly primed T cells. Although the cytokines necessary for several effector lineages have been identified, the innate cues that drive T follicular helper (Tfh) lineage cell development remain unclear. Here we found that following priming, CD4 T cells undergoing clonal expansion acquire a transient Tfh-like phenotype before differentiating into other effector lineages. In addition, we found that T cell-intrinsic myeloid differentiation antigen 88 (MyD88) signaling, which occurs downstream of interleukin-1 (IL-1) and IL-18 receptors, is critical for the primed CD4 T cells to transition out of the temporary Tfh lineage. Mice with T cell-specific deletion of MyD88 have a higher proportion of Tfh cells and germinal center (GC) B cells. These exaggerated Tfh cell and GC B cell responses, however, do not lead to protective immunity against infections. We demonstrate that T cell-intrinsic MyD88 is critical for effector lineage differentiation as well as production of the cytokines that are necessary for class switching. Overall, our study establishes that following priming and clonal expansion, CD4 T cells undergo a transitional Tfh-like phase and that further differentiation into effector lineages is dictated by T cell-intrinsic MyD88-dependent cues.

[Establishment of porcine model of prolonged cardiac arrest and cardiopulmonary resuscitation electrically induced by ventricular fibrillation].

OBJECTIVE: To investigate the optimal injury time point of cardiac arrest (CA) induced electrically, and establish a reproducible prolonged CA and cardiopulmonary resuscitation (CPR) model in pigs. METHODS: Forty healthy domestic male pigs were randomly divided into four groups, which were ventricular fibrillation (VF) 8, 10, 11, and 12 minutes groups, each group for 10 animals. In these groups, VF was induced by alternating current delivered to right ventricular endocardium and untreated for 8, 10, 11, and 12 minutes, respectively, followed by 6 minutes of CPR procedure. The resuscitation and survival outcomes were recorded. Hemodynamic parameters and arterial blood gases of animals after successful resuscitation were measured and recorded for 6 hours. Those successful resuscitation animals were regularly evaluated for the neurological deficit score (NDS) and survival outcomes every 24 hours till 96 hours after resuscitation. RESULTS: The shortest duration of CPR (minute: 6.9±1.3) and the highest successful ratio of the first defibrillation (7/10) were observed in group VF 8 minutes, and the ratio of successful resuscitation was 100%. The best coronary perfusion pressure (CPP) during the CPR, less neurological impairment, longer survival time, more stable hemodynamics, and shorter time for arterial pH and lactate level restoring to the original state after CPR were also observed in group VF 8 minutes, and no severe damage was found in those animals. The longest duration of CPR (minute: 10.3±2.9) and the lowest successful ratio of the first defibrillation (1/10) were observed in group VF 12 minutes, and only 4 animals achieved restoration of spontaneous circulation (ROSC), and no animal survived to CPR 96 hours. The worst CPP during CPR and the highest NDS after resuscitation were also found in VF 12 minutes animals compared to those animals in the other groups. The injuries caused by ischemia and hypoxia in groups VF 10 minutes and VF 11 minutes were in between those of the groups VF 8 minutes and VF 12 minutes, and the duration of CPR were (7.0±2.1) minutes and (8.2±2.6) minutes. There were 9 and 7 animals achieved ROSC in groups VF 10 minutes and VF 11 minutes correspondingly, and 6 and 4 animals survived to 96 hours respectively. Obviously unstable hemodynamics was observed during the period of CPR 2 hours in the two groups. At CPR 1 hour, the heart rates (HR, beats/min) in groups VF 10 minutes and VF 11 minutes increased to 172 (155, 201) and 168 (136, 196) respectively, and the mean arterial pressures (MAP, mmHg, 1 mmHg = 0.133 kPa) declined to 97 (92, 100) and 81 (77, 100), the cardiac output (CO, L/min) decreased to 5.0 (4.0, 5.8), 3.7 (3.0, 5.4) correspondingly. Distinct injuries were found in the two groups [CPR 24-96 hours NDS in groups VF 10 minutes and VF 11 minutes: 180 (110, 255)-20 (0, 400) and 275 (223, 350)-240 (110, 400)], and the arterial pH of the two group decreased to 7.26±0.09 and 7.23±0.09 respectively, and the level of lactate (mmol/L) increased to 9.17±1.48 and 12.80±2.71 correspondingly at CPR 0.5 hour. Significantly lower pH was observed in group VF 11 minutes compared to group VF 8 minutes at CPR 0.5 hour (7.23±0.09 vs. 7.33±0.04, P < 0.05). The highest level of lactate (mmol/L) was also found at the same time point in group VF 11 minutes, which recovered to normal slowly, and was still significantly higher than groups VF 8, 10, 12 minutes (7.58±3.99 vs. 2.55±1.53, 2.13±2.00, 3.40±2.30, all P < 0.05) at CPR 4 hours. CONCLUSIONS: The longer duration of CA was, the more severe damage would be, the longer CPR time would be required, and the harder of the animals to achieve ROSC. In this prolonged CA and CPR porcine model, 10-11 minutes for untreated VF, was an optimal time point with appropriate successful rate of resuscitation, survival outcomes, and post-resuscitation injuries. Therefore, we recommended 10-11 minutes might be the rational length of no-flow time in this model.

The putative Walker A and Walker B motifs of Rrp2 are required for the growth of Borrelia burgdorferi.

Rrp2 encodes a putative bacterial enhancer binding protein (bEBP) in Borrelia burgdorferi. Point mutation (G239C) of Rrp2 abolishes the transcriptional activation of σ54 -dependent rpoS. In contrast to canonical bEBPs that are dispensable for bacterial growth, Rrp2 is essential for borrelial growth in BSK medium. It has been believed that Rrp2's ATPase activity is not required for cell growth, but experimental evidence supporting this notion has been lacking. In particular, it has remained unclear whether the residue G239 is involved in Rrp2's presumptive ATPase activity. To address these information gaps, we examined the roles of Rrp2's potential strategic signatures including the G239 residue and the putative Walker A and Walker B motifs. Herein it was showed that Rrp2 has ATP binding and hydrolysis activities engendered by the Walker A and B motifs respectively. However, these activities were not significantly impaired by a G239C mutation. Further mutagenesis analyses indicated that Rrp2's Walker A and B motifs are required for borrelial growth; mutations of key residues in these two motifs were lethal to B. burgdorferi. The combined data suggest that the Walker A and Walker B motifs of Rrp2 are involved in the control of another unknown RpoS-independent gene product(s) associated with borrelial replication.

Evidence that BosR (BB0647) Is a Positive Autoregulator in Borrelia burgdorferi.

Borrelia burgdorferi survives in nature through a complex tick-mammalian life cycle. During its transit between ticks and mammalian hosts, B. burgdorferi must dramatically alter its outer surface profile in order to interact with and adapt to these two diverse niches. It has been established that the regulator BosR (BB0647) in B. burgdorferi plays important roles in modulating borrelial host adaptation. However, to date, how bosR expression itself is controlled in B. burgdorferi remains largely unknown. Previously, it has been shown that DNA sequences upstream of BosR harbor multiple sites for the binding of recombinant BosR, suggesting that BosR may influence its own expression in B. burgdorferi However, direct experimental evidence supporting this putative autoregulation of BosR has been lacking. Here, we investigated the expression of bosR throughout the tick-mammal life cycle of B. burgdorferi via quantitative reverse transcription (RT)-PCR analyses. Our data indicated that bosR is expressed not only during mouse infection, but also during the tick acquisition, intermolt, and transmission phases. Further investigation revealed that bosR expression in B. burgdorferi is influenced by environmental stimuli, such as temperature shift and pH change. By employing luciferase reporter assays, we also identified two promoters potentially driving bosR transcription. Our study offers strong support for the long-postulated function of BosR as an autoregulator in B. burgdorferi.

BadR (BB0693) controls growth phase-dependent induction of rpoS and bosR in Borrelia burgdorferi via recognizing TAAAATAT motifs.

In Borrelia burgdorferi (Bb), the alternative sigma factor RpoS plays a central role during Bb's adaptation to ticks and mammals. Previous studies have demonstrated that RpoS is not expressed during the early stages of spirochetal growth or when Bb resides in ticks during the intermolt phase, but the molecular details of these events remain unknown. In the current study, biomagnetic bead separation of rpoS promoter-binding proteins, coupled with genetic inactivation, was employed to identify BadR (BB0693) as a negative regulator that controls growth phase-dependent induction of rpoS and bosR in Bb. When badR was inactivated, the expression of rpoS and bosR was induced only during the early stages of bacterial growth, but not during the stationary growth phase. Recombinant BadR bound to the promoter DNA of rpoS and the regulatory region upstream of bosR via AT-rich TAAAATAT motifs. Mutations in this motif markedly inhibited or abolished rBadR binding. These results suggest that BadR directly influences the expression of both rpoS and bosR in Bb. This newly recognized role for BadR to fine-tune the activation of the RpoN-RpoS pathway at strategic times in Bb's life cycle potentially represents another layer of gene control over σ(54)-dependent gene regulation.

Synthesis of RpoS is dependent on a putative enhancer binding protein Rrp2 in Borrelia burgdorferi.

The RpoN-RpoS regulatory pathway plays a central role in governing adaptive changes by B. burgdorferi when the pathogen shuttles between its tick vector and mammalian hosts. In general, transcriptional activation of bacterial RpoN (σ54)-dependent genes requires an enhancer binding protein. B. burgdorferi encodes the putative enhancer binding protein Rrp2. Previous studies have revealed that the expression of σ54-dependent rpoS was abolished in an rrp2 point mutant. However, direct evidence linking the production of Rrp2 in B. burgdorferi and the expression of rpoS has been lacking, primarily due to the inability to inactivate rrp2 via deletion or insertion mutagenesis. Herein we introduced a regulatable (IPTG-inducible) rrp2 expression shuttle plasmid into B. burgdorferi, and found that the controlled up-regulation of Rrp2 resulted in the induction of σ54-dependent rpoS expression. Moreover, we created an rrp2 conditional lethal mutant in virulent B. burgdorferi. By exploiting this conditional mutant, we were able to experimentally manipulate the temporal level of Rrp2 expression in B. burgdorferi, and examine its direct impact on activation of the RpoN-RpoS regulatory pathway. Our data revealed that the synthesis of RpoS was coincident with the IPTG-induced Rrp2 levels in B. burgdorferi. In addition, the synthesis of OspC, a lipoprotein required by B. burgdorferi to establish mammalian infection, was rescued in the rrp2 point mutant when RpoS production was restored, suggesting that Rrp2 influences ospC expression indirectly via its control over RpoS. These data demonstrate that Rrp2 is required for the synthesis of RpoS, presumably via its action as an enhancer binding protein for the activation of RpoN and subsequent transcription of rpoS in B. burgdorferi.

Identification of a core sequence for the binding of BosR to the rpoS promoter region in Borrelia burgdorferi.

The alternative sigma factor RpoS in Borrelia burgdorferi plays a central role in modulating host adaptive responses when spirochaetes cycle between ticks and mammals. The transcriptional activation of σ(54)-dependent rpoS requires a Fur homologue designated BosR. Previously, BosR was shown to directly activate rpoS transcription by binding to the rpoS promoter. However, many other DNA binding features of BosR have remained obscure. In particular, the precise DNA sequence targeted by BosR has not yet been completely elucidated. The prediction of a putative Per box within the rpoS promoter region has further confounded the identification of the BosR binding sequence. Herein, by using electrophoretic mobility shift assays, we demonstrate that the putative Per box predicted in the rpoS promoter region is not involved in the binding of BosR. Rather, a 13 bp palindromic sequence (ATTTAANTTAAAT) with dyad symmetry, which we denote as the 'BosR box', functions as the core sequence recognized by BosR in the rpoS promoter region of Borrelia burgdorferi. Similar to a Fur box and a Per box, the BosR box probably comprises a 6-1-6 inverted repeat composed of two hexamers (ATTTAA) in a head-to-tail orientation. Selected mutations in the BosR box prevented recombinant BosR from binding to rpoS. In addition, we found that sequences neighbouring the BosR box also are required for the formation of BosR-DNA complexes. Identification of the BosR box advances our understanding of how BosR recognizes its DNA target(s), and provides new insight into the mechanistic details behind the unique regulatory function of BosR.