Borrelia burgdorferi , the causative agent of Lyme disease, can be recovered from different organs of infected animals and patients, indicating that the spirochete is very invasive. Motility and chemotaxis contribute to the invasiveness of B. burgdorferi and play important roles in the process of the disease. Recent reports have shown that motility is required for establishing infection in mammals. However, the role of chemotaxis in virulence remains elusive. Our previous studies showed that cheA ₂ , a gene encoding a histidine kinase, is essential for the chemotaxis of B. burgdorferi . In this report, the cheA ₂ gene was inactivated in a low-passage-number virulent strain of B. burgdorferi. In vitro analyses (microscopic observations, computer-based bacterial tracking analysis, swarm plate assays, and capillary tube assays) showed that the cheA ₂ mutant failed to reverse and constantly ran in one direction; the mutant was nonchemotactic to attractants. Mouse needle infection studies showed that the cheA ₂ mutant failed to infect either immunocompetent or immunodeficient mice and was quickly eliminated from the initial inoculation sites. Tick-mouse infection studies revealed that although the mutant was able to survive in ticks, it failed to establish a new infection in mice via tick bites. The altered phenotypes were completely restored when the mutant was complemented. Collectively, these data demonstrate that B. burgdorferi needs chemotaxis to establish mammalian infection and to accomplish its natural enzootic cycle.

Life cycle alternation between arthropod and mammals forces the Lyme disease spirochete, Borrelia burgdorferi , to adapt to different host milieus by utilizing diverse carbohydrates. Glycerol and chitobiose are abundantly present in the Ixodes tick. B. burgdorferi can utilize glycerol as a carbohydrate source for glycolysis and chitobiose to produce N -acetylglucosamine (GlcNAc), a key component of the bacterial cell wall. A recent study reported that Rrp1, a response regulator that synthesizes cyclic diguanylate (c-di-GMP), governs glycerol utilization in B. burgdorferi . In this report, we found that the rrp1 mutant had growth defects and formed membrane blebs that led to cell lysis when GlcNAc was replaced by chitobiose in the growth medium. The gene chbC encodes a key chitobiose transporter of B. burgdorferi . We found that the expression level of chbC was significantly repressed in the mutant and that constitutive expression of chbC in the mutant successfully rescued the growth defect, indicating a regulatory role of Rrp1 in chitobiose uptake. Immunoblotting and transcriptional studies revealed that Rrp1 is required for the activation of bosR and rpoS and that its impact on chbC is most likely mediated by the BosR-RpoS regulatory pathway. Tick-mouse infection studies showed that although the rrp1 mutant failed to establish infection in mice via tick bite, exogenous supplementation of GlcNAc into unfed ticks partially rescued the infection. The finding reported here provides us with new insight into the regulatory role of Rrp1 in carbohydrate utilization and virulence of B. burgdorferi .

The genome of Borrelia burgdorferi , the Lyme disease spirochete, encodes a homolog (the bb0184 gene product) of the carbon storage regulator A protein (CsrA _Bb ); recent studies reported that CsrA _Bb is involved in the regulation of several infectivity factors of B. burgdorferi . However, the mechanism involved remains unknown. In this report, a csrA _Bb mutant was constructed and complemented in an infectious B31A3 strain. Subsequent animal studies showed that the mutant failed to establish an infection in mice, highlighting that CsrA _Bb is required for the infectivity of B. burgdorferi . Western blot analyses revealed that the virulence-associated factors OspC, DbpB, and DbpA were attenuated in the csrA _Bb mutant. The Rrp2-RpoN-RpoS pathway (σ ⁵⁴ -σ ^S sigma factor cascade) is a central regulon that governs the expression of ospC , dbpB , and dbpA . Further analyses found that the level of RpoS was significantly decreased in the mutant, while the level of Rrp2 remained unchanged. A recent study reported that the overexpression of BB0589, a phosphate acetyl-transferase (Pta) that converts acetyl-phosphate to acetyl-coenzyme A (CoA), led to the inhibition of RpoS and OspC expression, suggesting that acetyl-phosphate is an activator of Rrp2. Along with this report, we found that CsrA _Bb binds to the leader sequence of the bb0589 transcript and that the intracellular level of acetyl-CoA in the csrA _Bb mutant was significantly increased compared to that of the wild type, suggesting that more acetyl-phosphate was being converted to acetyl-CoA in the mutant. Collectively, these results suggest that CsrA _Bb may influence the infectivity of B. burgdorferi via regulation of acetate metabolism and subsequent activation of the Rrp2-RpoN-RpoS pathway.

The Lyme disease spirochete Borrelia burgdorferi lacks the transcriptional cascade control of flagellar protein synthesis common to other bacteria. Instead, it relies on a post-transcriptional mechanism to control its flagellar synthesis. The underlying mechanism of this control remains elusive. A recent study reported that the increased level of BB0184 (CsrA_Bb; a homologue of carbon storage regulator A) substantially inhibited the accumulation of FlaB, the major flagellin protein of B. burgdorferi. In this report, we deciphered the regulatory role of CsrA_Bb on FlaB synthesis and the mechanism involved by analysing two mutants, csrA_Bb^- (a deletion mutant of csrA_Bb) and csrA_Bb⁺ (a mutant conditionally overexpressing csrA_Bb). We found that FlaB accumulation was significantly inhibited in csrA_Bb⁺ but was substantially increased in csrA_Bb^-. In contrast, the levels of other flagellar proteins remained unchanged. Cryo-electron tomography and immuno-fluorescence microscopic analyses revealed that the altered synthesis of CsrA_Bb in these two mutants specifically affected flagellar filament length. The leader sequence of flaB transcript contains two conserved CsrA-binding sites, with one of these sites overlapping the Shine–Dalgarno sequence. We found that CsrA_Bb bound to the flaB transcripts via these two binding sites, and this binding inhibited the synthesis of FlaB at the translational level. Taken together, our results indicate that CsrA_Bb specifically regulates the periplasmic flagellar synthesis by inhibiting translation initiation of the flaB transcript.

Viroporins are small, hydrophobic trans-membrane viral proteins that oligomerize to form hydrophilic pores in the host cell membranes. These proteins are crucial for the pathogenicity and replication of viruses as they aid in various stages of the viral life cycle, from genome uncoating to viral release. In addition, the ion channel activity of viroporin causes disruption in the cellular ion homeostasis, in particular the calcium ion. Fluctuation in the calcium level triggers the activation of the host defensive programmed cell death pathways as well as the inflammasome, which in turn are being subverted for the viruses’ replication benefits. This review article summarizes recent developments in the functional investigation of viroporins from various viruses and their contributions to viral replication and virulence.

Objective: HCV infection affects millions of people worldwide, and many patients develop chronic infection leading to liver cancers. For decades, the lack of a small animal model that can recapitulate HCV infection, its immunopathogenesis and disease progression has impeded the development of an effective vaccine and therapeutics. We aim to provide a humanised mouse model for the understanding of HCV-specific human immune responses and HCV-associated disease pathologies. Design: Recently, we have established human liver cells with a matched human immune system in NOD-scid Il2rg^−/− (NSG) mice (HIL mice). These mice are infected with HCV by intravenous injection, and the pathologies are investigated. Results: In this study, we demonstrate that HIL mouse is capable of supporting HCV infection and can present some of the clinical symptoms found in HCV-infected patients including hepatitis, robust virus-specific human immune cell and cytokine responses as well as liver fibrosis and cirrhosis. Similar to results obtained from the analysis of patient samples, the human immune cells, particularly T cells and macrophages, play critical roles during the HCV-associated liver disease development in the HIL mice. Furthermore, our model is demonstrated to be able to reproduce the therapeutic effects of human interferon alpha 2a antiviral treatment. Conclusions: The HIL mouse provides a model for the understanding of HCV-specific human immune responses and HCV-associated disease pathologies. It could also serve as a platform for antifibrosis and immune-modulatory drug testing.