A pleiotropic role of sialidase in the pathogenicity of Porphyromonas gingivalis.

As one of the keystone pathogens of periodontitis, the oral bacterium Porphyromonas gingivalis produces an array of virulence factors, including a recently identified sialidase (PG0352). Our previous report involving loss-of-function studies indicated that PG0352 plays an important role in the pathophysiology of P. gingivalis. However, this report had not been corroborated by gain-of-function studies or substantiated in different P. gingivalis strains. To fill these gaps, herein we first confirm the role of PG0352 in cell surface structures (e.g., capsule) and serum resistance using P. gingivalis W83 strain through genetic complementation and then recapitulate these studies using P. gingivalis ATCC33277 strain. We further investigate the role of PG0352 and its counterpart (PGN1608) in ATCC33277 in cell growth, biofilm formation, neutrophil killing, cell invasion, and P. gingivalis-induced inflammation. Our results indicate that PG0352 and PGN1608 are implicated in P. gingivalis cell surface structures, hydrophobicity, biofilm formation, resistance to complement and neutrophil killing, and host immune responses. Possible molecular mechanisms involved are also discussed. In summary, this report underscores the importance of sialidases in the pathophysiology of P. gingivalis and opens an avenue to elucidate their underlying molecular mechanisms.

A chemosensory-like histidine kinase is dispensable for chemotaxis in vitro but regulates the virulence of Borrelia burgdorferi through modulating the stability of RpoS.

As an enzootic pathogen, the Lyme disease bacterium Borrelia burgdorferi possesses multiple copies of chemotaxis proteins, including two chemotaxis histidine kinases (CHK), CheA1 and CheA2. Our previous study showed that CheA2 is a genuine CHK that is required for chemotaxis; however, the role of CheA1 remains mysterious. This report first compares the structural features that differentiate CheA1 and CheA2 and then provides evidence to show that CheA1 is an atypical CHK that controls the virulence of B. burgdorferi through modulating the stability of RpoS, a key transcriptional regulator of the spirochete. First, microscopic analyses using green-fluorescence-protein (GFP) tags reveal that CheA1 has a unique and dynamic cellular localization. Second, loss-of-function studies indicate that CheA1 is not required for chemotaxis in vitro despite sharing a high sequence and structural similarity to its counterparts from other bacteria. Third, mouse infection studies using needle inoculations show that a deletion mutant of CheA1 (cheA1mut) is able to establish systemic infection in immune-deficient mice but fails to do so in immune-competent mice albeit the mutant can survive at the inoculation site for up to 28 days. Tick and mouse infection studies further demonstrate that CheA1 is dispensable for tick colonization and acquisition but essential for tick transmission. Lastly, mechanistic studies combining immunoblotting, protein turnover, mutagenesis, and RNA-seq analyses reveal that depletion of CheA1 affects RpoS stability, leading to reduced expression of several RpoS-regulated virulence factors (i.e., OspC, BBK32, and DbpA), likely due to dysregulated clpX and lon protease expression. Bulk RNA-seq analysis of infected mouse skin tissues further show that cheA1mut fails to elicit mouse tnf-α, il-10, il-1ß, and ccl2 expression, four important cytokines for Lyme disease development and B. burgdorferi transmigration. Collectively, these results reveal a unique role and regulatory mechanism of CheA1 in modulating virulence factor expression and add new insights into understanding the regulatory network of B. burgdorferi.

Functional and structural analyses reveal that a dual domain sialidase protects bacteria from complement killing through desialylation of complement factors.

The complement system is the first line of innate immune defense against microbial infections. To survive in humans and cause infections, bacterial pathogens have developed sophisticated mechanisms to subvert the complement-mediated bactericidal activity. There are reports that sialidases, also known as neuraminidases, are implicated in bacterial complement resistance; however, its underlying molecular mechanism remains elusive. Several complement proteins (e.g., C1q, C4, and C5) and regulators (e.g., factor H and C4bp) are modified by various sialoglycans (glycans with terminal sialic acids), which are essential for their functions. This report provides both functional and structural evidence that bacterial sialidases can disarm the complement system via desialylating key complement proteins and regulators. The oral bacterium Porphyromonas gingivalis, a "keystone" pathogen of periodontitis, produces a dual domain sialidase (PG0352). Biochemical analyses reveal that PG0352 can desialylate human serum and complement factors and thus protect bacteria from serum killing. Structural analyses show that PG0352 contains a N-terminal carbohydrate-binding module (CBM) and a C-terminal sialidase domain that exhibits a canonical six-bladed ß-propeller sialidase fold with each blade composed of 3-4 antiparallel ß-strands. Follow-up functional studies show that PG0352 forms monomers and is active in a broad range of pH. While PG0352 can remove both N-acetylneuraminic acid (Neu5Ac) and N-glycolyl-neuraminic acid (Neu5Gc), it has a higher affinity to Neu5Ac, the most abundant sialic acid in humans. Structural and functional analyses further demonstrate that the CBM binds to carbohydrates and serum glycoproteins. The results shown in this report provide new insights into understanding the role of sialidases in bacterial virulence and open a new avenue to investigate the molecular mechanisms of bacterial complement resistance.

Chemotaxis Coupling Protein CheW2 Is Not Required for the Chemotaxis but Contributes to the Full Pathogenicity of Borreliella burgdorferi.

The bacterial chemotaxis regulatory circuit mainly consists of coupling protein CheW, sensor histidine kinase CheA, and response regulator CheY. Most bacteria, such as Escherichia coli, have a single gene encoding each of these proteins. Interestingly, the Lyme disease pathogen, Borreliella burgdorferi, has multiple chemotaxis proteins, e.g., two CheA, three CheW, and three CheY proteins. The genes encoding these proteins mainly reside in two operons: cheW2-cheA1-cheB2-cheY2 (A-I) and cheA2-cheW3-cheX-cheY3 (A-II). Previous studies demonstrate that all the genes in A-II are essential for the chemotaxis of B. burgdorferi; however, the role of those genes in A-I remains unknown. This study aimed to fill this gap using the CheW2 gene, the first gene in A-I, as a surrogate. We first mapped the transcription start site of A-I upstream of cheW2 and identified a σ70-like promoter (PW2) and two binding sites (BS1 and BS2) of BosR, an unorthodox Fur/Per homolog. We then demonstrated that BosR binds to PW2 via BS1 and BS2 and that deletion of bosR significantly represses the expression of cheW2 and other genes in A-I, implying that BosR is a positive regulator of A-I. Deletion of cheW2 has no impact on the chemotaxis of B. burgdorferi in vitro but abrogates its ability to evade host adaptive immunity, because the mutant can establish systemic infection only in SCID mice and not in immunocompetent BALB/c mice. This report substantiates the previous proposition that A-I is not implicated in chemotaxis; rather, it may function as a signaling transduction pathway to regulate B. burgdorferi virulence gene expression.

Phosphoproteomics Unravel HBV Triggered Rewiring of Host Phosphosignaling Events.

Hepatitis B virus (HBV) infection persists as a major global health problem despite the availability of HBV vaccines for disease prevention. However, vaccination rates remains low in some regions of the world, driving the need for novel strategies to minimise infections and prevent disease progression. Thus, understanding of perturbed molecular signaling events during early phases of HBV infection is required. Phosphosignaling is known to be involved in the HBV infection processes, yet systems-level changes in phosphosignaling pathways in the host during infection remain unclear. To this end, we performed phosphoproteome profiling on HBV-infected HepG2-NTCP cells. Our results showed that HBV infection drastically altered the host phosphoproteome and its associated proteins, including kinases. Computational analysis of this phosphoproteome revealed dysregulation of the pathways involved in immune responses, cell cycle processes, and RNA processing during HBV infection. Kinase Substrate Enrichment Analysis (KSEA) identified the dysregulated activities of important kinases, including those from CMGC (CDK, MAPK, GSK, and CLK), AGC (protein kinase A, G, and C), and TK (Tyrosine Kinase) families. Of note, the inhibition of CLKs significantly reduced HBV infection in HepG2-NTCP cells. In all, our study unravelled the aberrated phosphosignaling pathways and the associated kinases, presenting potential entry points for developing novel therapeutic strategies for HBV treatment.

Hepatitis C virus mediated chronic inflammation and tumorigenesis in the humanised immune system and liver mouse model.

Hepatitis C is a liver disease caused by infection of the Hepatitis C virus (HCV). Many individuals infected by the virus are unable to resolve the viral infection and develop chronic hepatitis, which can lead to formation of liver cirrhosis and cancer. To understand better how initial HCV infections progress to chronic liver diseases, we characterised the long term pathogenic effects of HCV infections with the use of a humanised mouse model (HIL mice) we have previously established. Although HCV RNA could be detected in infected mice up to 9 weeks post infection, HCV infected mice developed increased incidences of liver fibrosis, granulomatous inflammation and tumour formation in the form of hepatocellular adenomas or hepatocellular carcinomas by 28 weeks post infection compared to uninfected mice. We also demonstrated that chronic liver inflammation in HCV infected mice was mediated by the human immune system, particularly by monocytes/macrophages and T cells which exhibited exhaustion phenotypes. In conclusion, HIL mice can recapitulate some of the clinical symptoms such as chronic inflammation, immune cell exhaustion and tumorigenesis seen in HCV patients. Our findings also suggest that persistence of HCV-associated liver disease appear to require initial infections of HCV and immune responses but not long term HCV viraemia.

Characterisation of liver pathogenesis, human immune responses and drug testing in a humanised mouse model of HCV infection.

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.

Viral Membrane Channels: Role and Function in the Virus Life Cycle.

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.

NS5B induces up-regulation of the BH3-only protein, BIK, essential for the hepatitis C virus RNA replication and viral release.

Hepatitis C virus (HCV) induces cytopathic effects in the form of hepatocytes apoptosis thought to be resulted from the interaction between viral proteins and host factors. Using pathway specific PCR array, we identified 9 apoptosis-related genes that are dysregulated during HCV infection, of which the BH3-only pro-apoptotic Bcl-2 family protein, BIK, was consistently up-regulated at the mRNA and protein levels. Depletion of BIK protected host cells from HCV-induced caspase-3/7 activation but not the inhibitory effect of HCV on cell viability. Furthermore, viral RNA replication and release were significantly suppressed in BIK-depleted cells and over-expression of the RNA-dependent RNA polymerase, NS5B, was able to induce BIK expression. Immunofluorescence and co-immunoprecipitation assays showed co-localization and interaction of BIK and NS5B, suggesting that BIK may be interacting with the HCV replication complex through NS5B. These results imply that BIK is essential for HCV replication and that NS5B is able to induce BIK expression.

Study of the response regulator Rrp1 reveals its regulatory role in chitobiose utilization and virulence of Borrelia burgdorferi.

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.

Borrelia burgdorferi needs chemotaxis to establish infection in mammals and to accomplish its enzootic cycle.

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 cheA2, a gene encoding a histidine kinase, is essential for the chemotaxis of B. burgdorferi. In this report, the cheA2 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 cheA2 mutant failed to reverse and constantly ran in one direction; the mutant was nonchemotactic to attractants. Mouse needle infection studies showed that the cheA2 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.

Carbon storage regulator A (CsrA(Bb)) is a repressor of Borrelia burgdorferi flagellin protein FlaB.

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.

Inactivation of bb0184, which encodes carbon storage regulator A, represses the infectivity of Borrelia 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.