Cui Hua Liu

An Immunomodulatory Molecule of Symbiotic Bacteria Directs Maturation of the Host Immune System

The mammalian gastrointestinal tract harbors a complex ecosystem consisting of countless bacteria in homeostasis with the host immune system. Shaped by evolution, this partnership has potential for symbiotic benefit. However, the identities of bacterial molecules mediating symbiosis remain undefined. Here we show that, during colonization of animals with the ubiquitous gut microorganism Bacteroides fragilis, a bacterial polysaccharide (PSA) directs the cellular and physical maturation of the developing immune system. Comparison with germ-free animals reveals that the immunomodulatory activities of PSA during B. fragilis colonization include correcting systemic T cell deficiencies and T(H)1/T(H)2 imbalances and directing lymphoid organogenesis. A PSA mutant of B. fragilis does not restore these immunologic functions. PSA presented by intestinal dendritic cells activates CD4+ T cells and elicits appropriate cytokine production. These findings provide a molecular basis for host-bacterial symbiosis and reveal the archetypal molecule of commensal bacteria that mediates development of the host immune system.

Regulation of surface architecture by symbiotic bacteria mediates host colonization

Microbes occupy countless ecological niches in nature. Sometimes these environments may be on or within another organism, as is the case in both microbial infections and symbiosis of mammals. Unlike pathogens that establish opportunistic infections, hundreds of human commensal bacterial species establish a lifelong cohabitation with their hosts. Although many virulence factors of infectious bacteria have been described, the molecular mechanisms used during beneficial host–symbiont colonization remain almost entirely unknown. The novel identification of multiple surface polysaccharides in the important human symbiont Bacteroides fragilis raised the critical question of how these molecules contribute to commensalism. To understand the function of the bacterial capsule during symbiotic colonization of mammals, we generated B. fragilis strains deleted in the global regulator of polysaccharide expression and isolated mutants with defects in capsule expression. Surprisingly, attempts to completely eliminate capsule production are not tolerated by the microorganism, which displays growth deficits and subsequent reversion to express capsular polysaccharides. We identify an alternative pathway by which B. fragilis is able to reestablish capsule production and modulate expression of surface structures. Most importantly, mutants expressing single, defined surface polysaccharides are defective for intestinal colonization compared with bacteria expressing a complete polysaccharide repertoire. Restoring the expression of multiple capsular polysaccharides rescues the inability of mutants to compete for commensalism. These findings suggest a model whereby display of multiple capsular polysaccharides provides essential functions for bacterial colonization during host–symbiont mutualism.

Characteristics and Treatment Outcomes of Patients with MDR and XDR Tuberculosis in a TB Referral Hospital in Beijing: A 13-Year Experience

Background Information on treatment outcomes among hospitalized patients with multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) are scarce in China. Methodology/Principal Findings We conducted this retrospective study to analyze the characteristics and treatment outcomes in MDR- and XDR-TB patients in the 309 Hospital in Beijing, China during 1996–2009. Socio-demographic and clinical data were retrieved from medical records and analyzed. Logistic regression analysis was performed to identify risk factors associated with poor treatment outcomes and Cox proportional hazards regression model was further used to determine risk factors associated with death in TB patients. Among the 3,551 non-repetitive hospitalized TB patients who had drug susceptibility testing (DST) results, 716 (20.2%) had MDR-TB and 51 (1.4%) had XDR-TB. A total of 3,270 patients who had medical records available were used for further analyses. Treatment success rates (cured and treatment completed) were 90.9%, 53.4% and 29.2% for patients with non-MDR-TB, patients with MDR-TB excluding XDR-TB and patients with XDR-TB, respectively. Independent risk factors associated with poor treatment outcomes in MDR-TB patients included being a migrant (adjusted OR = 1.77), smear-positivity at treatment onset (adjusted OR = 1.94) and not receiving 3 or more potentially effective drugs (adjusted OR = 3.87). Independent risk factors associated with poor treatment outcomes in XDR-TB patients were smear-positivity at treatment onset (adjusted OR = 10.42) and not receiving 3 or more potentially effective drugs (adjusted OR = 14.90). The independent risk factors associated with death in TB patients were having chronic obstructive pulmonary disease (adjusted HR = 5.25) and having hypertension (adjusted HR = 4.31). Conclusions/Significance While overall satisfactory treatment success for non-MDR-TB patients was achieved, more intensive efforts should be made to better manage MDR- and XDR-TB cases in order to improve their treatment outcomes and to minimize further emergence of so-called totally drug-resistant TB cases.

Mycobacterium tuberculosis suppresses innate immunity by coopting the host ubiquitin system

Mycobacterium tuberculosis PtpA, a secreted tyrosine phosphatase essential for tuberculosis pathogenicity, could be an ideal target for a drug against tuberculosis, but its active-site inhibitors lack selectivity over human phosphatases. Here we found that PtpA suppressed innate immunity dependent on pathways of the kinases Jnk and p38 and the transcription factor NF-κB by exploiting host ubiquitin. Binding of PtpA to ubiquitin via a region with no homology to human proteins activated it to dephosphorylate phosphorylated Jnk and p38, leading to suppression of innate immunity. Furthermore, the host adaptor TAB3 mediated NF-κB signaling by sensing ubiquitin chains, and PtpA blocked this process by competitively binding the ubiquitin-interacting domain of TAB3. Our findings reveal how pathogens subvert innate immunity by coopting host ubiquitin and suggest a potential tuberculosis treatment via targeting of ubiquitin-PtpA interfaces.

Insights into battles between Mycobacterium tuberculosis and macrophages

ABSTRACTAs the first line of immune defense for Mycobacterium tuberculosis (Mtb), macrophages also provide a major habitat for Mtb to reside in the host for years. The battles between Mtb and macrophages have been constant since ancient times. Triggered upon Mtb infection, multiple cellular pathways in macrophages are activated to initiate a tailored immune response toward the invading pathogen and regulate the cellular fates of the host as well. Toll-like receptors (TLRs) expressed on macrophages can recognize pathogen-associated-molecular patterns (PAMPs) on Mtb and mediate the production of immune-regulatory cytokines such as tumor necrosis factor (TNF) and type I Interferons (IFNs). In addition, Vitamin D receptor (VDR) and Vitamin D-1-hydroxylase are up-regulated in Mtb-infected macrophages, by which Vitamin D participates in innate immune responses. The signaling pathways that involve TNF, type I IFNs and Vitamin D are inter-connected, which play critical roles in the regulation of necroptosis, apoptosis, and autophagy of the infected macrophages. This review article summarizes current knowledge about the interactions between Mtb and macrophages, focusing on cellular fates of the Mtb-infected macrophages and the regulatory molecules and cellular pathways involved in those processes.

Genotypic Analysis of Klebsiella pneumoniae Isolates in a Beijing Hospital Reveals High Genetic Diversity and Clonal Population Structure of Drug-Resistant Isolates

Background The genetic diversity and the clinical relevance of the drug-resistant Klebsiella pneumoniae isolates from hospital settings are largely unknown. We thus conducted this prospective study to analyze the molecular epidemiology of K. pneumoniae isolates from patients being treated in the 306 Hospital in Beijing, China for the period of November 1, 2010–October 31, 2011. Methodology/Principal Findings Antibiotic susceptibility testing, PCR amplification and sequencing of the drug resistance-associated genes, and multilocus sequence typing (MLST) were conducted. A total of 163 isolates were analyzed. The percentage of MDR, XDR and PDR isolates were 63.8% (104), 20.9 (34), and 1.8% (3), respectively. MLST results showed that 60 sequence types (STs) were identified, which were further separated by eBURST into 13 clonal complexes and 18 singletons. The most dominant ST was ST15 (10.4%). Seven new alleles and 24 new STs were first identified in this study. Multiple logistic regression analysis revealed that certain clinical characteristics were associated with those prevalent STs such as: from ICU, from medical ward, from community acquired infection, from patients without heart disease, from patients with treatment success, susceptible to extended spectrum cephalosporin, susceptible to cephamycins, susceptible to fluoroquinolones, and with MDR. Conclusions/Significance Our data indicate that certain drug-resistant K. pneumoniae clones are highly prevalent and are associated with certain clinical characteristics in hospital settings. Our study provides evidence demonstrating that intensive nosocomial infection control measures are urgently needed.

Existence and characterization of allelic variants of Sao, a newly identified surface protein from Streptococcus suis

Abstract Surface antigen one (Sao) is a newly identified protein from the major zoonotic pathogen, Streptococcus suis. In search of functional proteins related to the pathogenesis of Chinese S. suis 2 (SS2), unexpectedly, a variant of Sao protein was obtained. To test its prevalence in S. suis, PCR assay was adopted to address the coding genes systematically. It was found that there are three allelic variants of sao gene, namely sao-S, sao-M, and sao-L based on the different lengths of the genes (∼1.5, ∼1.7, and ∼2.0 kb, respectively). These differences were determined to be caused by heterogeneity within the number of C-terminal repeat sequences (R), which had been seen as a pathogenicity-related domain in the plant pathogen, Xanthomonas oryzae. Two variants (sao-M and sao-L) were only found in SS2. All three variant proteins were prepared in vitro and their biochemical and biophysical properties were characterized. A soluble form of Sao-M protein was then used as a capture antigen to develop an enzyme-linked immunosorbent assay method to detect antibodies against SS2 in convalescent pig sera. Taken together, the results exhibit the properties of Sao proteins and provide an efficient Sao-M-based method for monitoring SS2 infection.

Analysis of Drug Resistance Determinants in Klebsiella pneumoniae Isolates from a Tertiary-Care Hospital in Beijing, China

Background The rates of multidrug-resistant (MDR), extensively drug-resistant (XDR) and pandrug-resistant (PDR) isolates among Enterobacteriaceae isolates, particularly Klebsiella pneumoniae, have risen substantially worldwide. Methodology/Principal Findings To better understand the molecular mechanisms of drug resistance in K. pneumoniae, we analyzed the drug resistance determinants for K. pneumoniae isolates collected from the 306 Hospital, a tertiary-care hospital in Beijing, China, for the period of September 1, 2010-October 31, 2011. Drug susceptibility testing, PCR amplification and sequencing of the drug resistance determinants were performed. Conjugation experiments were conducted to examine the natural ability of drug resistance to disseminate among Enterobacteriaceae strains using a sodium azide-resistant Escherichia coli J53 strain as a recipient. Among the 223 consecutive non-repetitive K. pneumoniae isolates included in this study, 101 (45.3%) were extended-spectrum beta-lactamases (ESBLs) positive. The rates of MDR, XDR, and PDR isolates were 61.4% (n = 137), 22.0% (n = 49), and 1.8% (n = 4), respectively. Among the tested drug resistance-associated genes, the following ones were detected at relatively high rates bla CTX-M-10 (80, 35.9%), aacC2 (73, 32.7%), dhfr (62, 27.8%), qnrS (58, 26.0%), aacA4 (57, 25.6%), aadA1 (56, 25.1%). Results from conjugation experiments indicate that many of the drug resistance genes were transmissible. Conclusions/Significance Our data give a “snapshot” of the complex genetic background responsible for drug resistance in K. pneumoniae in China and demonstrate that a high degree of awareness and monitoring of those drug resistance determinants are urgently needed in order to better control the emergence and transmission of drug-resistant K. pneumoniae isolates in hospital settings.

Comparative genomic analysis of Mycobacterium tuberculosis clinical isolates

BackgroundDue to excessive antibiotic use, drug-resistant Mycobacterium tuberculosis has become a serious public health threat and a major obstacle to disease control in many countries. To better understand the evolution of drug-resistant M. tuberculosis strains, we performed whole genome sequencing for 7 M. tuberculosis clinical isolates with different antibiotic resistance profiles and conducted comparative genomic analysis of gene variations among them.ResultsWe observed that all 7 M. tuberculosis clinical isolates with different levels of drug resistance harbored similar numbers of SNPs, ranging from 1409–1464. The numbers of insertion/deletions (Indels) identified in the 7 isolates were also similar, ranging from 56 to 101. A total of 39 types of mutations were identified in drug resistance-associated loci, including 14 previously reported ones and 25 newly identified ones. Sixteen of the identified large Indels spanned PE-PPE-PGRS genes, which represents a major source of antigenic variability. Aside from SNPs and Indels, a CRISPR locus with varied spacers was observed in all 7 clinical isolates, suggesting that they might play an important role in plasticity of the M. tuberculosis genome. The nucleotide diversity (Л value) and selection intensity (dN/dS value) of the whole genome sequences of the 7 isolates were similar. The dN/dS values were less than 1 for all 7 isolates (range from 0.608885 to 0.637365), supporting the notion that M. tuberculosis genomes undergo purifying selection. The Л values and dN/dS values were comparable between drug-susceptible and drug-resistant strains.ConclusionsIn this study, we show that clinical M. tuberculosis isolates exhibit distinct variations in terms of the distribution of SNP, Indels, CRISPR-cas locus, as well as the nucleotide diversity and selection intensity, but there are no generalizable differences between drug-susceptible and drug-resistant isolates on the genomic scale. Our study provides evidence strengthening the notion that the evolution of drug resistance among clinical M. tuberculosis isolates is clearly a complex and diversified process.

The two-component system Ihk/Irr contributes to the virulence of Streptococcus suis serotype 2 strain 05ZYH33 through alteration of the bacterial cell metabolism.

Streptococcus suis serotype 2 (S. suis 2) is an important zoonotic pathogen. It causes heavy economic losses in the pig-farming industry and can be associated with severe infections in humans, e.g. streptococcal toxic shock syndrome. Understanding its pathogenesis is critical for prevention and control of diseases caused by S. suis 2. In this study, we show that deletion of a two-component system (TCS), 05SSU1660/1661 (orthologues of the Ihk/Irr TCS of Streptococcus pyogenes), in S. suis 2 strain 05ZYH33 results in notable attenuation of virulence, as exemplified by reduced adherence to mucosal epithelium cells, increased elimination by macrophages, reduced ability to survive in an acidic or oxidative-stressed environment, and lowered pathogenicity in mice. Further analysis of differential proteomics profiles by two-dimensional electrophoresis revealed that while many previously well-known virulence factors, such as suilysin, autolysin and muraminidase-released protein, were not expressed differentially, cell metabolism was downregulated in the Ihk/Irr deletion mutant. In addition, the oxidative-stress response gene for manganese-dependent superoxide dismutase (MnSOD) was also repressed significantly in the mutant. Collectively, our data suggest that the Ihk/Irr TCS contributes to the virulence of S. suis 2 strain 05ZYH33, mainly through alteration of the bacterial cell metabolism.