Richard Kao

The University of Hong Kong | HKU · Department of Microbiology

Influenza viruses (IAV) continue to pose an imminent threat to human due to annual influenza epidemics outbreak and episodic pandemics with high mortality. In this context, the suboptimal vaccine coverage and efficacy, coupled with recurrent events of viral resistance against a very limited antiviral portfolio, emphasize an urgent need for new addi...

Recently, we showed that Mp1p is an important virulence factor of Talaromyces marneffei, a dimorphic fungus phylogenetically closely related to Aspergillus fumigatus. In this study, we investigated the virulence properties of the four Mp1p homologues (Afmp1p, Afmp2p, Afmp3p, and Afmp4p) in A. fumigatus using a mouse model. All mice died 7 days afte...

The PB1 C-terminal domain and PB2 N-terminal domain interaction of the influenza A polymerase, which modulates the assembly of PB1 and PB2 subunits, may serve as a valuable target for the development of novel anti-influenza therapeutics. In this study, we performed a systematic screening of a chemical library, followed by the antiviral evaluation o...

As antibiotic resistance becomes phenomenal, alternative therapeutic strategies for bacterial infections such as anti-virulence treatments have been advocated. We have constructed a total of 20 gfp-luxABCDE dual-reporter plasmids with selected promoters from S. aureus virulence-associated genes. The plasmids were introduced into various S. aureus s...

Background: Talaromyces marneffei is an opportunistic dimorphic fungus prevalent in Southeast Asia. We previously demonstrated that Mp1p is an immunogenic surface and secretory mannoprotein of T. marneffei. Since Mp1p is a surface protein that can generate protective immunity, we hypothesized that Mp1p and/or its homologs are virulence factors. M...

Analysis of transcript formation of the Mp1p homologs under the two culture conditions. The constitutively expressed actin was used as control. (TIF)

ELISA detecting the expression of Mp1p. Diluted culture supernatants of wild-type T. marneffei strain PM1, MP1 knockout mutant and MP1 complemented mutant were used for Mp1p detection in ELISA. Culture medium was used as control. (TIF)

Primers used in this study. (DOC)

Southern blot of genomic DNA from the wild-type T. marneffei strain PM1 and the MP1 knockout mutant. The genomic DNA was digested with SpeI and probed with 1-kb MP1 upstream region probe and MP1 probe. Homologous recombination of the deletion construct at the MP1 locus resulted in integration of the hygromycin resistance gene that increased the siz...

Western blot showing expression of Mp1p. Lane 1, Wild-type T. marneffei strain PM1. Lane 2, MP1 knockout mutant. Lane 3, MP1 complemented mutant. (TIF)

PCR and agarose gel electrophoresis detecting the presence/absence of MP1. Lane 1, Wild-type T. marneffei strain PM1. Lane 2, MP1 knockout mutant. Lane 3, MP1 complemented mutant. Lane 4, MP1 knockdown mutant. (TIF)

Influenza viruses are among the most common pathogens that threaten the health of humans and animals worldwide. Various anti-viral therapeutic agents are currently used for treatment and prophylaxis of influenza virus, but the targets of these drugs are easily mutated and result in resistance. Therefore, medications that have broad spectrum coverag...

The hemagglutinin (HA) protein of influenza A virus initiates cell entry by binding to sialic acids on target cells. In the current study, we demonstrated that in addition to sialic acids, influenza A/Puerto Rico/8/34 H1N1 (PR8) virus HA specifically binds to cell surface nucleolin (NCL). The interaction between HA and NCL was initially revealed wi...

Objectives The conserved residues 318–483 in the PB2 subunit of influenza A polymerase is an independently folded cap-binding domain (PB2cap) that exhibits a distinct binding mode from other host cap-binding proteins, which suggests that PB2cap might be an ideal drug target. This study aimed to identify a new class of anti-influenza inhibitors that...

Immunomodulators have been shown to improve the outcome of severe pneumonia. We have previously shown that mycophenolic acid (MPA), an immunomodulator, has antiviral activity against influenza A/WSN/1933(H1N1) using a high-throughput chemical screening assay. This study further investigated the antiviral activity and mechanism of action of MPA agai...

The RNA-dependent RNA polymerase of influenza A virus comprises conserved and independently-folded subdomains with defined functionalities. The N-terminal domain of the PA subunit (PAN) harbors the endonuclease function so that it can serve as a desired target for drug discovery. To identify a class of anti-influenza inhibitors that impedes PAN end...

To prevent the attachment of bacteria to implant surfaces, the 1D zinc oxide nanowire-coating has been successfully developed on material surfaces by using a custom-made hydrothermal approach. The chemical nature, surface topography and wettability of spike-like 1D ZnO nanowire-coating are comprehensively investigated. The anti-adhesive and antimic...

Ultra-selective and ultra-sensitive probes for hypochlorous acid (HOCl), one of the most poorly understood reactive oxygen species (ROS), are urgently needed to unravel the HOCl functions in important biological processes such as development and innate immunity. Based on a selective oxidative O-dearylation reaction of 2,6-dichlorophenol toward HOCl...

Phorbol esters, which are protein kinase C (PKC) activators, and histone deacetylase (HDAC) inhibitors, which cause enhanced acetylation of cellular proteins, are the main classes of chemical inducers of Epstein-Barr virus (EBV) lytic cycle in latently EBV-infected cells acting through the PKC pathway. Chemical inducers which induce EBV lytic cycle...

Expression of various lytic proteins induced by the hit compounds in HONE1-EBV and YCCEL1 cells. HONE1-EBV cells or YCCEL1 cells were treated with the hit compounds at their optimal concentration to induce lytic cycle. The expression of various EBV lytic proteins was detected at different time points post-treatment. Compound E11 consistently induce...

Activation of the cellular kinase pathways by romidepsin and compound E11. AGS-BX1 cells were treated with romidepsin (R) at 5nM for 24h or E11 at 20μM at the specified time points. Romidepsin treatment increased phosphorylation of PKCδ and ATM but not JNK, while vice versa for E11. (TIF)

Tertiary screening in NA cells. (a) Expression of EBV immediately-early (IE) lytic proteins, Zta, Rta, and early protein EA-D (BMRF1) in NA cells 48h post-treatment by the top 40 compounds in tertiary screening. The concentrations used were the approximate half inhibitory concentration (IC50) for cell proliferation. The 22 compounds with an asteris...

Lytic induction kinetics of the hit compounds at early periods of treatment. AGS-BX1 cells were treated with the hit compounds at various time points to observe for the time point in which increase in expression of the viral IE protein Zta was first detected. Compound E11 and C7 is the fastest to induce lytic cycle, with the increase in Zta express...

Rotterlin, a specific PKCδ inhibitor, inhibited lytic induction by the HDAC inhibitor SAHA. HONE1-EBV cells were pre-treated with specific inhibitors of PI3K (LY294002, 15 μM), MEK (PD98059, 50μM), JNK (SP600125, 50μM), p38 MAPK (SB202190, 20μM) and PKCδ (Rottlerin, 10μM) for 1h before the addition of 10μM SAHA. Cells were harvest after 48h for exa...

Enhanced induction of EBV lytic cycle by the hit compounds and the HDAC inhibitor SAHA. AGS-BX1 cells were treated with 2.5μM of SAHA and various concentrations of E11 or C7 for 24h. Expression of viral IE protein Zta was detected to by western blotting to estimate the magnitude of lytic induction. The combinations with an asterisk (*) are the conc...

Importance: Here we report two structurally distinctive novel fusion inhibitors of influenza A virus that act by interfering with the structural change of HA at acidic pH, a process necessary for successful entry of the virus. Mutational and molecular docking studies have identified their binding pockets closely situated to the B-loop region of he...

Staphylococcus aureus is a common pathogen found in the community and in hospitals. Most notably, methicillin-resistant S. aureus is resistant to many antibiotics, which is a growing public health concern. The emergence of drug-resistant strains has prompted the search for alternative treatments, such as immunotherapeutic approaches. To date, most...

Assembly of the heterotrimeric influenza virus polymerase complex from the individual subunits PB1, PA, and PB2 is a prerequisite for viral replication, in which the interaction between the C terminal of PA (PAC) and the N-terminal of PB1 (PB1N) may be a desired target for antiviral development. In this study, we compared the feasibility of high th...

Peroxiredoxin I (Prx I) plays an important role in cancer development and inflammation. It is a dual-functional protein which acts as both an antioxidant enzyme and a molecular chaperone. While there have been intensive studies on its peroxidase activity, Prx I's chaperone activity remains elusive, likely due to the lack of chaperone inhibitors. He...

Although the major causes of isoniazid (INH) resistance in Mycobacterium tuberculosis are confined to structural mutations in katG and promoter mutations in the mabA-inhA operon, a significant proportion of INH-resistant strains have unknown resistance mechanisms. Recently, we identified a high-level INH-resistant M. tuberculosis clinical isolate,...

Objective: Candida infections are major problem in immunocompromised populations. The limited number of antifungals and emergence of drug-resistant strains have posed a huge clinical challenge and highlighted the dire need for novel antifungal agents with new mechanism of action. Recently, a high-throughput screening of a library with 50,240 small...

Candida is the most common fungal pathogen of humans worldwide and has become a major clinical problem because of the growing number of immunocompromised patients, who are susceptible to infection. Moreover, the number of available antifungals is limited, and antifungal-resistant Candida strains are emerging. New and effective antifungals are there...

Background: Surfactant proteins play a key role in alveolar stability. We examined whether single nucleotide polymorphisms (SNPs) related to the surfactant protein genes are associated with severe influenza. Methods: In the first cohort, 12 SNPs related to surfactant protein genes were compared between Chinese patients with severe and mild pande...

Middle East respiratory syndrome coronavirus (MERS-CoV) has emerged to cause fatal infections in patients in the Middle East and traveler-associated secondary cases in Europe and Africa. Person-to-person transmission is evident in outbreaks involving household and hospital contacts. Effective antivirals are urgently needed. We used small compound-b...

Objective: Candida is the main causative agent of denture stomatitis (DS) which affects up to 67% denture wearers. Treatment of DS with existing topical antifungals is challenging due to effect of saliva and short contact time. Emergence of antifungal resistance has resulted in frequent treatment failure. Therefore, new antifungal with steady-relea...

Penicillium marneffei is an opportunistic fungal pathogen endemic in Southeast Asia, causing lethal systemic infections in immunocompromised patients. P. marneffei grows in a mycelial form at the ambient temperature of 25°C and transitions to a yeast form at 37°C. The ability to alternate between the mycelial and yeast forms at different temperatur...

Novel antivirals are needed to supplement existing control strategies for influenza A virus (IAV). A promising new class of drug, exemplified by the compound nucleozin, has recently been identified that targets the viral nucleoprotein (NP). These inhibitors are thought to act as “molecular staples” that stabilize interactions between NP monomers, p...

Objective: Candida infections is a major clinical problem worldwide due to growing body of compromised populations. Current antifungal agents suffer from various limitations including emergence of drug resistance strains and systemic side effects. Therefore, in the present study, we evaluated the novel lead compound SM21 and its functional analogue...

Penicillium marneffei is the most important thermal dimorphic, pathogenic fungus endemic in China and Southeast Asia and is particularly important in HIV-positive patients. We report the 28,887,485-bp draft genome sequence of P. marneffei, which contains its complete mitochondrial genome, sexual cycle genes, a high diversity of Mp1p homologues, and...

Objective: Candida albicans (Ca) is the major fungal pathogen in humans which causes both superficial mucosal and systemic mycoses. Emergence of drug resistant Ca in the past decade highlights the dire need of novel antifungal agents. In the present study, we characterized antifungal activity of a novel small molecule using oral and systemic candid...

Severe pandemic influenza A H1N1 (2009) infection, especially in the lower respiratory tract, is often associated with the virus carrying a D222G substitution in the hemagglutinin (HA) protein of the virus. The mechanism for this association has not been fully explored. In the in vitro binding assay, it was found that clinical isolates carrying D22...

The surface compatibility and antibacterial properties of biomaterials are crucial to tissue engineering and other medical applications, and plasma-assisted technologies have been employed to enhance these characteristics with good success. Herein, we describe and review the recent developments made by our interdisciplinary team on self-antimicrobi...

Prunus mume is a common fruit in Asia, which has been used in traditional Chinese medicine. In this study, we focused on the antimicrobial properties of Prunus mume extract against oral pathogens related to dental caries and periodontal diseases. A total of 15 oral pathogens including Streptococcus mutans, S. sobrinus, S. mitis, S. sanguinis, Lacto...

Protein transduction domains (PTD) have been identified to have the capacity to facilitate molecular cargo to translocate through cell membrane. This study aims to utilize the cell membrane penetrating ability of octaarginine oligopeptide, a simplified prototype of the PTD, to enhance the transfection efficiency of chitosan. Octaarginine-modified c...

the aim of this study was to evaluate the antimicrobial efficacy of Traditional Chinese Medicine Fructus mume on a monospecies-biofilm model established on orthodontic brackets in vitro. the antimicrobial effect of Fructus mume aqueous extract on the planktonic Streptococcus mutans (S. mutans) was tested by microdilution method (MIC). The cell viab...

alpha-Sarcin, mitogillin, and restrictocin are small (approximately 17 kDa) basic robosome-inactivating proteins (RIPs) produced by the Aspergilli that catalytically inactivate the large ribosomal subunits of all organisms tested to date. These three fungal ribotoxins act as specific ribonucleases by hydrolyzing one single phosphodiester bond in th...

Modified strontium-containing hydroxyapatite (Sr-HA) bone cement was loaded with gentamicin sulfate to generate an efficient bioactive antibiotic drug delivery system for treatment of bone defects. Gentamicin release and its antibacterial property were determined by fluorometric method and inhibition of Staphylococcus aureus (S. aureus) growth. Gen...

A PCR-sequencing assay was evaluated for direct detection of mutations in the quinolone resistance-determining region (QRDR) of gyrase A (gyrA) gene in fluoroquinolone-resistant Mycobacterium tuberculosis in respiratory specimens. As determined by gyrA QRDR analysis, complete concordance of genotypic and phenotypic fluoroquinolone resistance was de...

Influenza A remains a significant public health challenge because of the emergence of antigenically shifted or highly virulent strains. Antiviral resistance to available drugs such as adamantanes or neuraminidase inhibitors has appeared rapidly, creating a need for new antiviral targets and new drugs for influenza virus infections. Using forward ch...

Twenty traditional Chinese medicines (TCM) were evaluated for their antimicrobial activity against four common oral bacteria. TCMs were tested for sensitivity against Streptococcus mitis, Streptococcus sanguis, Streptococcus mutans and Porphyromonas gingivalis. Aliquots of suspension of each bacterial species were inoculated onto a horse blood agar...

To assess whether vancomycin MIC creeps among blood methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from 5 hospitals in Hong Kong from 1997 to 2008. Blood cultures MRSA isolates from 1997 to 1999 (period 1), 2004 (period 2) and 2006-2008 (period 3) were retrieved. Etest method was used to determine their vancomycin MIC. The ge...

The spike (S) protein of SARS coronavirus (SARS-CoV) has been known to recognize and bind to host receptors, whose conformational changes then facilitate fusion between the viral envelope and host cell membrane, leading to viral entry into target cells. However, other functions of SARS-CoV S protein such as proteolytic cleavage and its implications...

SARS-coronavirus (SARS-CoV) encodes a main protease, 3CLpro, which plays an essential role in the viral life cycle and is currently the prime target for discovering new anti-coronavirus agents. In this article, we report our success in developing a novel red-shifted (RS) fluorescence-based assay for 3CLpro and its application for identifying small-...

Multiplex allele-specific PCRs detecting katG codon 315 and mabA (bp −15) mutations could specifically identify 77.5% of isoniazid-resistant Mycobacterium tuberculosis strains in the South China region. One clinical isolate harboring InhA Ile194Thr was characterized to show strong association with isoniazid resistance in Mycobacterium tuberculosis.

RNase U2 is an endoribonuclease secreted by the fungus Ustilago sphaerogena. Its genomic DNA (rnu2), containing an intron of 116 bp, has been isolated and cloned. The corresponding cDNA has also been synthesized. The recombinant RNase U2 was successfully produced in Pichia pastoris, fused to the yeast alkaline phosphatase signal peptide. The recomb...

An Escherichia coli-Laribacter hongkongensis shuttle vector (pPW380) was constructed by ligating the 4701-bp EcoRI digested fragment of pHLHK8 to EcoRI digested pBK-CMV. An E. coli-L. hongkongensis inducible expression shuttle vector was further constructed by ligating a 2105-bp fragment that contains the tetracycline repressor and tetracycline-ind...

A 10-mer overlapping peptide library has been synthesized for screening and identification of linear B-cell epitopes of severe acute respiratory syndrome associated coronavirus (SARS-CoV), which spanned the major structural proteins of SARS-CoV. One hundred and eleven candidate peptides were positive according to the result of PEPscan, which were a...

The use of recombinant severe acute respiratory syndrome-coronavirus (SARS-CoV) nucleocapsid protein (N) enzyme-linked immunosorbent assay (ELISA)-based antibody and antigen tests for diagnosis of SARS-CoV infections have been widely reported. However, no recombinant SARS-CoV spike protein (S)-based ELISA is currently available. In this article, we...

Severe acute respiratory syndrome associated coronavirus main protease (SARS-CoV Mpro) has been proposed as a prime target for anti-SARS drug development. We have cloned and overexpressed the SARS-CoV Mpro in Escherichia coli, and purified the recombinant Mpro to homogeneity. The kinetic parameters of the recombinant SARS-CoV Mpro were characterize...