K. pneumoniae infections - Klebsiella pneumoniae

1.1 Klebsiella pneumoniae

1.1.1 K. pneumoniae infections

K. pneumoniae has emerged as the leading cause of pyogenic liver abscess in Asia,

with over 900 cases reported in Taiwan by 2004 [4]. Smaller series have been reported from other countries in Asia, including the Peoples Republic of China, Korea, Japan, Singapore, Hong Kong, Thailand and India [5]. Diabetes mellitus or impaired glucose tolerance is significant comorbidity, present in 40–75 % of these cases. A significant minority of patients (8–15%) had infection at other anatomical sites, including brain abscess, endophthalmitis, pyogenic meningitis, empyema, septic pulmonary emboli, osteomyelitis, septic arthritis, prostatic and psoas abscesses.

Mortality ranged from 4 to 11 %, which is notably lower than historical mortality rates reported for pyogenic liver abscesses [6,7]. In 1999, K. pneumoniae was first reported as a cause of community- acquired, mono-microbial pyogenic liver abscess

in North America [6,8]. In Europe, single cases have been reported from Spain, Belgium, the Netherlands, Sweden, Italy and France [9-13]. One larger case series from France published in 2011 detailed seven cases of invasive liver abscess from hospital [14]. In addition, it was identified as the commonest cause (23/79 cases) of pyogenic liver abscess at two New York City hospitals, between 1993 and 2003 [15].

Invasive isolates typically belong to capsular serotypes K1 and K2, both of which express a distinct hypermucoviscous phenotype [16]. Heavy encapsulation confers the bacteria resistance to phagocytosis and prevent from intracellular killing. Two plasmid-encoded virulence factors have been well characterized, rmpA, a regulator of mucoid phenotype that upregulates the capsule synthesis, and the iron siderophore aerobactin, which enables the bacterium to obtain iron. Other virulence factors include the genes kfu, which codes for an iron uptake system, and allS, which is associated with allantoin metabolism. All have been associated with severe pyogenic infections [17,18].

In the hospital environment with the extensive use of antibiotics, multiple drug resistance has been increasingly observed in K. pneumoniae, especially the extended-spectrum β-lactamase (ESBL)-producing strains [19-21]. Carbapenems are considered to be the preferred agents for the treatment of serious infections caused by ESBL-producing K. pneumoniae because of their high stability against β-lactamase

hydrolysis and observed retained susceptibility of ESBL producers [22-24]. Nerveless, it is rare occurrence for liver abscesses caused by ESBL- K pneumoniae have been reported in Taiwan. Carbapenem-resistant K pneumoniae, such as strains producing NDM-1, has been increasingly reported [25,26]. Antibiotics such as ampicillin–sulbactam, a third-generation cephalosporin, aztreonam, and a quinolone can be used t treat these hyper-resistance strains [27].

1.2 Klebsiella pneumoniae virulence factors

The virulence factors that allow K. pneumoniae to overcome innate host immunity and to maintain infection in a mammalian host factors include capsule, lipopolysaccharides, adhesins, iron acquisition systems, serum resistance factor, and biofilm formation regulators [28,29]. CPS acts to protect the bacteria from phagocytosis, from killing by polymorphonuclear granulocytes and from killing by bactericidal serum factors [30-32]. Besides the physical hindrance to fimbrial binding, the role of Klebsiella CPS in mediating the bacterial resistance to antimicrobial peptides has also been reported [33,34]. K. pneumoniae strains expressing K1 and K2 CPS are the most virulent to mice [35]. In addition to the 77 serotypes distinguished, a new K serotype has been identified in 2008 [36]. Serotypes K1, K2, K4 and K5 are highly virulent in experimental infection in mice and are often associated with severe infections in humans and animals [37]. The K1 and K2 serotypes were also found to

be the most prevalent capsular serotypes in liver abscess-causing K. pneumoniae. The rmpA (regulator of the mucoid phenotype A) gene correlated with abscess formation

in patients with community-acquired K. pneumoniae bacteremia has been attributed to be a risk factor for metastatic infection in patients with K. pneumoniae liver abscess [38]. The rmpA together with rmpA2 gene both located on the large virulence plasmid pLVPK are able to enhance the CPS biosynthesis thereby confer K. pneumoniae a hypermucoviscosity phenotype [39].

Animal experiments have been performed to assess the role of iron-acquisition systems in K. pneumoniae pathogenicity [40,41]. Analysis of the genomic sequence of K. pneumoniae NTUH-K2044 revealed 10 putative iron-acquisition systems, whereas

K. pneumoniae strain MGH78578 and CG43 possess only 6 and 8 of these systems,

respectively [42].

The LPS O-antigen and the lipid A are responsible for the resistance to serum factors and the establishment of septic shock [43]. Antimicrobial peptides, such as polymyxin B, are bactericidal agents that exert their effects by interacting with the LPS of Gram-negative bacteria. Our previous studied have shown that regulation of the gene expression for LPS modification determine the polymyxin B resistance [44,45]. The adhesion factors, including the type 1 [46,47] and 3 fimbriae [48] play crucial roles in adhesion to host cells, persistence in infection site, and biofilm

formation.

1.2.1 K. pneumoniae type 1 fimbriae

Type 1 fimbriae are approximately 7 nm wide and 1-2 μm long surface organelles are found in many bacteria in the family Enterobacteriaceae [49,50]. Type 1 fimbriae are encoded by fimAICDEFGH gene cluster coding for the fimbrial structure and assembly. The fimbrial rod consists of the major subunits FimA and the minor subunits FimI, FimF, and FimG. The adhesive properties of type 1 fimbriae are exerted by the FimH adhesin which locates at the tips of the fimbriae. FimC and FimD are respectively chaperone and usher that are required for the fimbrial assembly.

[51,52]. Type 1 fimbriae, which are able to cause mannose-sensitive agglutination of yeast cells or erythrocytes (mannose-sensitive haemagglutination, MSHA) from guinea pig are regulated via phase- variation [47,51,53-57].

The fimK gene, locating downstream to the fimH gene and unique to the K.

pneumoniae fim gene cluster, encodes an EAL domain protein [56]. We have recently

demonstrated that FimK may influence type 1 fimbriation by binding to fimS via the N-terminal domain, and thereafter, the altered protein structure activates C-terminal PDE activity to reduce the intracellular c-di-GMP level[58].

1.2.2 K. pneumoniae type 3 fimbriae

Type 3 fimbriae that are originally characterized in Klebsiella strains are 2-4 nm

wide and 0.5-2 μm long surface organelles. The fimbriae are able to agglutinate the tannic acid-treated human erythrocytes which could not be competitively inhibited by mannose and hence designated MR/K haemagglutination [59]. Besides playing an important role in biofilm formation on biotic and abiotic surfaces, type 3 fimbriae are able to attach the endothelial and bladder epithelial cell lines [60-65]. The fimbriae are encoded by chromosomally- or plasmid-borne mrkABCDF [61,65,66]. MrkD is the adhesin that mediates binding specificity and biofilm formation on extracellular matrix-coated surfaces , which can bind to type IV and/or type V collagen [63].

1.3 Cyclic-di-GMP signaling system

In bacteria, there are having various ways to sense environmental signals and to adapt their behavior and physiology through different signaling transduction systems.

In bacteria, there are various ways to sense environmental signals and to adapt their behavior and physiology through different signaling transduction systems. Cyclic AMP (cAMP) is a global second messenger involved in bacterial transcription regulation, while the signaling role of cyclic di-GMP (c-di-GMP) which was first discovered in G. xylinus [67] as an allosteric activator of the cellulose synthesis has not been recognized until recently. The second messenger c-di-GMP exclusively found in bacteria is involved in many fundamental behaviors such as motility, sessility and virulence. It also plays key roles in adhesion to surfaces, aggregation and

biofilm formation, and developmental transitions [68,69] [70].

1.3.1 The GGDEF- and EAL-domain proteins

In general, GGDEF domains are approximately 170 amino acids long and GG(D/E)EF motif is an integral part of the active site [71]. Stand-alone GGDEF domains are usually not enzymatically active, but require activation through an N-terminal signaling domain for activation. Besides the EAL motif, there are several other highly conserved motifs involved in catalysis, substrate binding and divalent ion coordination. Usually, EAL domains show significant enzymatic activity without N-terminal allosteric activation. Both GGDEF and EAL domain containing proteins can be enzymatically active [72-74]. Alternatively, only one domain possesses enzymatic activity, while the enzymatically inactive domain serves a regulatory function [75].

GGDEF or EAL domain proteins often contain additional sensory and signal transduction domains such as PAS, GAF, HAMP REC, and HTH domains [69,76,77].

It has been shown that oxygen, amino acids, electrons, and photons can modify the activity of DGC or PDE proteins. For example, PAS is a conserved protein domain involved in sensing oxygen, redox or light [78]; PleD is a GGDEF domain protein that carries the REC domain that gets phosphorylated to activate the DGC activity for c-di-GMP synthesis [79].

Many GGDEF domain proteins possess c-di-GMP diguanyl cyclase (DGC) activity, while EAL or HD-GYP domain proteins exert c-di-GMP phosphodiesterase (PDE) activity [72,80,81]. The concentration of c-di-GMP is modulated through the action of DGC and PDE respectively to synthesize and hydrolysis of c-di-GMP [82,83].

1.3.2 Regulatory mechanism of c-di-GMP

Through binding to diverse receptors or effectors, the c-di-GMP exerts a regulatory activity. The small ‘effector’ called PilZ domain, transcription factor or riboswitch [84-86]. The c-di-GMP-mediated regulation can occur at the level of transcription, post-transcription or post-translation, such as in the allosteric effect on cellulose synthesis or regulation of protein turnover [70]. In general, c-di-GMP is involved in positive regulation of exopolysaccharide production, biosynthesis of adhesive fimbriae and biofilm formation [58,82,87-93]. By contrast, motility is commonly negatively regulated by c-di-GMP. Various types of motility, including flagella-mediated swimming and swarming, type IV pili-mediated twitching motility are all affected by c-di-GMP [87,94]. In S. typhimurium, high levels of c-di-GMP generated by overexpression of the DGC AdrA inhibited swarming and swimming motility while reduction of c-di-GMP levels by overexpression of the PDE YhjH stimulated motility [95-97]. The binding of c-di-GMP to the PilZ domain protein

YcgR leads to a conformational change in the protein and consequently the c-di-GMP loaded YcgR form a complex with FliG and FliM to impair the flagella activity [85,98,99].

1.3.3 Role of c-di-GMP in virulence

C-di-GMP signaling is involved in regulating virulence of many bacteria which include E. coli, S. Typhimurium, Vibrio cholerae, Pseudomonas aeruginosa, Bordetella pertussis, Xanthomonas campestris, Legionella pneumophila, Brucella

melitensis and Anaplasma phagocytophilum [75,93,98,100-105]. In V. cholera,

downregulation of c-di-GMP levels by the PDE VieA leads to activation of cholera toxin [106]. P. aeruginosa c-di-GMP signaling is required for biofilm formation in chronic infection and also the acute infection phenotype [90,107,108]. The plant pathogen X. campestris pathovar campestris (Xcc) causes disease through a HD-GYP domain protein expression to regulate the production of extracellular enzymes and extracellular polysaccharide[109], and motility. In S. typhimurium, the EAL-domain like protein STM1344 causes resistance to oxidative stress, inhibits rapid macrophage killing and is required for virulence in the typhoid fever mouse model. Intriguingly, STM1344 has no PDE activity nor has bind activity to c-di-GMP, and hence the involvement of c-di-GMP signalling in these phenotypes remains to be investigated [110].

1.3.4 Domain structure of GGDEF- and EAL-protein encoding genes in K.

pneumoniae CG43S3

GGDEF and EAL domain proteins are widespread in bacterial genomes. Very often one bacterial genome contains more than one GGDEF and EAL domain protein.

The sequenced genome of S. typhimurium codes for 20 GGDEF/EAL domain proteins;

5 contain a GGDEF, 8 an EAL domain and 7 contain both. On the other hand, E. coli K-12 has 12 GGDEF, 12 EAL and 7 GGDEF-EAL domain proteins [111]. A recent study search for conserved GGDEF and EAL domains in three sequenced K.

pneumoniae genomes revealed multiple copies of GGDEF and EAL containing

proteins: 21 for K. pneumoniae Kp342, 18 for K. pneumoniae MGH 78578 and 17 for K. pneumoniae NTUH-K2044 [112]. The K. pneumoniae CG43 genome sequence has

recently been resolved and the annotation results show 24 GGDEF- and EAL-domain protein encoding genes. As shown in Fig. 1.1, 10 GGDEF-, 11 EAL- and 4 GGDEF-EAL-domain protein encoding genes are identified. The sensor domains identified include MASE, CHASE, CACHE and CSS motif [113]. Genome-wide approach to study these proteins might shed light on their functional roles in different environmental settings.

Fig. 1.1. Domain architecture of putative c-di-GMP signaling proteins encoded

by the K. pneumoniae CG43. The genome of K. pneumoniae CG43 encoding (A)

GGDEF, (B) EAL, and (C) GGDEF/EAL, the analysis of protein functional domain,

as indicated, was performed using the Pfam database provided online (http://www.sanger.ac.uk /Software/Pfam/). The identities of the proteins are also shown in blue: four EAL domain proteins (YjcC, FimK and MrkJ) that have been described in K. pneumoniae [58,91,114,115] and (chapter 2).

1.4 Oxidative stress

The most commonly discussed oxidants that cause damage to DNA, proteins, and cell membranes and often results in cell death are the reactive oxygen species (ROS) including superoxide anion (O₂^.-), hydrogen peroxide (H₂O₂), and the hydroxyl radical (HO^.), and the reactive nitrogen species (RNS), which include nitric oxide (NO^.) and peroxynitrite (ONOO^-). During infection, pathogens have equipped to protect themselves from the oxidative burst of phagocytic cells and the challenging oxidative environments within cellular and extracellular compartments.

The oxygen species can be excluded from active sites by electron transfer to the redox cofactors. Reactions of this type occur by the formation of reactive oxygen species (ROS) and their subsequent inactivation of enzymes. Intracellular molecular oxygen can adventitiously abstract electrons from the exposed redox moieties of electron-transfer enzymes, thereby generating partially reduced oxygen species. A mixture of O2⁻ and H₂O₂is formed, reflecting the fact that either one or two electrons can be transferred in an oxidation event. Flavoenzymes, which is ubiquitous and

abundant, in particular are responsible for all aerobic organisms to experience a steady flux of endogenously generated oxidants [116]. The overall reaction rate is proportional to collision frequency; thus, O2⁻ and H₂O₂ fluxes depend directly upon the ambient concentration of oxygen [117,118]. Several source of oxidative stress have been identified that include (a) intracellular enzyme autooxidation. In exponentially growing E.coli, both O₂^.-and H₂O₂ are generated by the auto-oxidation of components of the respiratory chain [119]. (b) envirmental redox reactions, (c) H₂O₂ released by competing microbes, (d) phagosomal NADPH oxidase and (e) redox cycling antibiotics , plants or microorganisms secrete redox-cycling antibiotics that diffuse into the competing bacteria, chemically oxidize redox enzymes and transfer the electrons to molecular oxygen [118].

1.4.1 Oxidative stress response in bacteria

The defense mechanisms, which play an important role in determining the bacterial virulence, include sensing, avoiding, and removing the oxidants. In Escherichia coli, superoxide is removed by SODs (SodA, SodB, SodC) [120],

generating hydrogen peroxide which is then removed by catalases (KatE, KatG) and peroxidases (AhpC). The transcriptome analysis of Pseudomonas aeruginosa or Staphylococcus aureus response to H₂O₂ revealed many more genes including the

virulence genes, the genes encoding products involved in DNA repair and anaerobic metabolism were induced. Nevertheless, many of the genome-wide analysis revealed the expression of more than 100 genes were induced in responding to either H₂O₂ or paraquat indicating the complexity of the antioxidant strategies [121-123].

Many of the defenses are controlled by regulators that respond to iron (e.g., Fur), oxygen tension (e.g., FNR and ArcAB), superoxide (e.g., SoxRS), and hydrogen peroxide (e.g., OxyR). In E. coli, OxyR is known to regulate more than nine genes including katG (hydroperoxidase I), ahpC (alkylhydroperoxide reductase), gorA (gluthione reductase), oxyS (regulatory RNA), and fur that are involved directly or indirectly in the oxidative stress response. The antioxidant genes katE, katG, and sodC have been reported to be components of RpoS regulon .While the expression of

superoxide dismutase SodA and SodB are respectively controlled by at least 3 global regulators including SoxRS, ArcAB, and Fur [124].

RpoS is a sigma factor which regulates expression of a variety of genes in both E.

coli and Salmonella spp. that allows the bacteria to adapt, resist, and survive under

stress condition. The deletion of rpoS in V. vulnificus was found to down-regulate the expression of fur, indicating a cascade regulation between the two global regulators.

Fur, which is complexed with iron as an iron responsive regulator, binds to multiple sites (a 19-bp Fur box) with differential affinities to repress the transcription of genes

required for iron acquisition, acid and oxidative stress responses. However, Fur has also been demonstrated to function as a transcriptional activator for several genes including fur itself (Hp and Vv), and virulence associated genes [125-127].

In K. pneumoniae CG43, the 2CS response regulator encoding gene kvgA deletion was found to reduce the expression of katG and sodC. KvgAS has also been shown to be induced expression in upon treatment with 0.2 mM paraquat, which suggesting an involvement of the 2CS in oxidative stress defense in the bacteria. Moreover, we have reported in K. pneumoniae CG43 that rpoS deletion reduced the kvgAS expression which also showing a cascade regulation on the expression of 2CS. This suggests complex signaling networks with inter-connection regulatory circuits are required for multiple stress signal integration [128].

1.5 Transcriptome analysis

Transcriptome analysis of gene expression can be profiled by high throughput techniques including SAGE, serial analysis of gene expression [129], microarray [130], and sequencing of clones from cDNA libraries [131]. Microarrays have been the method of choice providing high throughput and affordable costs. However, the microarray technology has limitations including insufficient sensitivity for quantifying lower abundant transcripts, narrow dynamic range and non-specific hybridizations.

Sequencing-based methods such as SAGE rely upon cloning and sequencing cDNA fragments. This approach allows quantification of mRNA abundance by counting the number of times cDNA fragments from a corresponding transcript are represented in a given sample, assuming that cDNA fragments sequenced contain sufficient information to identify a transcript. The currently used RNAseq is a SAGE-based technology that allows for high-throughput analysis of transcriptomes. Its decrease in the cost allows routinely used in research, increasing application and fast development.

In RNAseq method, complementary DNAs (cDNAs) generated from the mRNA of interest are directly sequenced using next-generation sequencing (NGS) technologies.

The obtained reads can then be aligned to a reference genome for a whole-genome transcriptome map.

The NGS technology enables massive parallel sequencing at a reasonable cost [132,133]. Current NGS platforms include the Roche 454 Genome Sequencer, Illumina's Genome Analyzer, and Applied Biosystems' SOLiD. These platforms can analyze tens to hundreds of millions of DNA fragments simultaneously, generate giga-bases of sequence information from a single run, and have revolutionized SAGE and cDNA sequencing technology [134].

1.5.1 RPKM measure

Measures of RNA abundance are important for many areas of biology and often obtained from RNAseq using Illumina sequence data. These measures need to be normalized to remove technical biases in the sequencing approach, such as the length of the RNA species and the sequencing depth of a sample. RPKM, which is the most frequently used measure of RNAseq data, calculates the number of reads mapped to a particular gene region (rg) and the feature length, flg [135]. The calculation is as RPKMg = rg x 10⁹/flg x R, where R is the total number of reads from the sequencing run of that sample.

Chapter 2 YjcC, a c-di-GMP phosphodiesterase protein, regulates the oxidative stress response and virulence of Klebsiella

pneumoniae CG43

2.1. Abstract

This study shows that the expression of yjcC, an in vivo expression (IVE) gene, and the stress response regulatory genes soxR, soxS, and rpoS are paraquat inducible in Klebsiella pneumoniae CG43. The deletion of rpoS or soxRS decreased yjcC expression, implying an RpoS- or SoxRS-dependent control. After paraquat or H2O2

treatment, the deletion of yjcC reduced bacterial survival. These effects could be complemented by introducing the ΔyjcC mutant with the YjcC-expression plasmid pJR1. The recombinant protein containing only the YjcC-EAL domain exhibited phosphodiesterase (PDE) activity; overexpression of yjcC has lower levels of cyclic di-GMP. The yjcC deletion mutant also exhibited increased reactive oxygen species (ROS) formation, oxidation damage, and oxidative stress scavenging activity. In addition, the yjcC deletion reduced capsular polysaccharide production in the bacteria, but increased the LD50 in mice, biofilm formation, and type 3 fimbriae major pilin MrkA production. Finally, a comparative transcriptome analysis showed 34 upregulated and 29 downregulated genes with the increased production of YjcC. The activated gene products include glutaredoxin I, thioredoxin, heat shock proteins, chaperone, and MrkHI, and proteins for energy metabolism (transporters, cell surface structure, and transcriptional regulation). In conclusion, the results of this study suggest that YjcC positively regulates the oxidative stress response and mouse

virulence but negatively affects the biofilm formation and type 3 fimbriae expression by altering the c-di-GMP levels after receiving oxidative stress signaling inputs.

2.2. Introduction

During infection, pathogens protect themselves from the oxidative burst of phagocytic cells and the challenging oxidative environments within cellular and extracellular compartments. Upon exposure to oxidative stress such as tellurite, paraquat or hydrogen peroxide, E. coli exhibits an increase in the intracellular ROS and the content of protein carbonyl groups [136-138]. Reactive oxygen species (ROS), including superoxide anion (O₂^.-), hydrogen peroxide (H₂O₂), and hydroxyl radicals (HO^.), may damage DNA, proteins, and cell membranes and often lead to cell death [117,118]. The bacterial defense mechanism includes sensing, avoiding, and

在文檔中克雷白氏肺炎桿菌CG43磷酸二酯酶YjcC與二級訊息分子c-di-GMP在壓力反應調控之功能性研究 (頁 16-0)