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國立交通大學

生物科技研究所

碩士論文

克雷白氏肺炎桿菌中纖毛表現之分析

Study on the fimbrial expression in

Klebsiella pneumoniae

研究生:廖朝陽

指導教授:彭慧玲 博士

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致謝

兩年的碩士班生涯就此告一段落,回憶過去的種種,無論在生活中或是研究 工作上都讓我學習到許多;感謝身邊的人一直以來的照顧及支持,讓我有所成長。 特別是我的指導老師彭慧玲博士,在研究上給我很大的發揮空間,也提供許 多寶貴的想法與意見。一直以來,給您增添了不少的麻煩;像是過去的許多報告、 對於一些觀念的謬思,或是這次的論文,老師都不辭辛勞、費心費力地給予教導, 在此致上我最深的感激。此外,這篇論文的完成,亦要感謝中興大學的黃秀珍老 師以及交通大學的林志生老師細心地指正與建議,讓此篇論文能夠更加完整。 感謝實驗室的夥伴們,豐富了我的碩士班生活。特別是盈蓉學姊耐心的指 導,讓我學習到許多實驗上的技巧;而健誠學長亦提供許多研究上的資訊以及充 分的討論,讓我收穫不少。此外,也要感謝格維和登魁這兩位實驗室的同學一路 上共同分享喜憂,伴隨著我一路成長;祝福你們都有美好的前程。 還有靜柔學姊、育聖學長、智凱學長、靖婷學姊、秉熹、雲龍、嘉怡、佳瑩、 振宇、純珊,謝謝這段時間的幫忙與包容,讓此求學期間增添了許多美好的回憶。 最後要感謝爸爸、媽媽、大姐、二姐以及我的好室友仲翔,謝謝您們給我鼓舞與 照顧,僅次論文獻給我的家人以及各位,謝謝您們。

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Contents

Page Contents………..………... Ⅰ Table content……….………..……… Ⅲ Figure content………..……….………….. Ⅳ Abbreviation……….………... Ⅵ Abstract in Chinese………..……….…... 1 Abstract……….……….……... 3 Introduction………..……….………... 5

Materials and Methods……….……….……… 15

Results Part Ι: Expressional analyses of the putative fimbriae in Klebsiella pneumoniae... 23

Part ΙΙ: Characterization of the putative regulators encoding by the genes respectively located in the vicinity to kpb, kpd, or kpf gene cluster……... 28

Discussion………..………….. 33

Reference.……….….……….…... 36

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Table

Page

Table 1. Bacterial strains and plasmids used in this study……….….. 46

Table 2. Primers used in this study………..…. 48

Table 3. Measurements of the fimbrial promoter activity on different

culture conditions………...… 49

Table 4. Measurements of the fimbrial promoter activity on different carbon

sources and bovine serum additions………... 50

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Figure

Page

Fig. 1. Organization of the putative fimbrial gene clusters in K.pneumoniae

NTUH-K2044………...………..………...…...… 52

Fig. 2. Promoter activity measurement in K. pneumoniae CG43S3Z01 and CG43S3Z01phgS-………...………....… 53

Fig. 3. Sequence alignment of PhgS with other representative members of MarR family………...….………....………... 54

Fig. 4. Nucleotide sequence of PkpgA……… 55

Fig. 5. Nucleotide sequence of PmrkA………...………. 56

Fig. 6. Activity measurements of PkpgA, PkpgA1, PkpgA2, PmrkA, and Pmrkt…...…. 57

Fig. 7. Schematic representation of the entericidin loci....……….…...…. 58

Fig. 8. Organization of kpbRABCD and the BLAST result of KpbR... 59

Fig. 9. Over-expression of KpbR in E. coli JM109….………...……….. 60

Fig. 10. Over-expression of KpdR in K. pneumoniae and the organization of kpdRABCD………. 63

Fig. 11. PCR detection of blaTEM-116 in K. pneumoniae NTUH-K2044 and CG43S3………..……….... 64

Fig. 12. Ampicillin susceptibility assay……….………... 65

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Fig. 14. Biofilm formation capability... 68

Fig. 15. Effect of KpdR over-expression on the capsular synthesis………….…… 69

Fig. 16. Organization of kpfABCD and kpfR genes, and the BLAST result of

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Abbreviation

BLAST basic local alignment search tool

BCIP 5-bromo-4-chloro-3-indolyl phosphate

ESBL extended-spectrum beta-lactamase

2CS two component system

kDa kiloDalton

AFA a-fimbrial adhesins

NFA non-fimbrial adhesins

CPS capsular polysaccharide

PG peptidoglycan

IPTG isopropyl-1-thio-β-D-galactopyranoside

LB Luria-Bertani

mRNA messenger RNA

NBT nitro blue tetrazolium chloride

ONPG ο-nitrophenyl-β-D-galactopyranoside PCR polymerase chain reaction

PAGE polyacrylamide gel electrophoresis

(9)

Lrp leucine-responsive regulatory protein

IHF integration host factor

CAP catabolite gene activator protein

H-NS histone-like protein

Dam deoxyadenosine methylase

TFA trifluoroacetic acid

(10)

中文摘要

克雷白氏肺炎桿菌是伺機性感染的革蘭氏陰性菌。利用生物資訊分析方法 HMMER,我們在一株從台灣大學附設醫院的病患身上所分離出具有高致病性的 NTUH-K0244 菌株的基因體中找到九套線毛基因組。除了已被廣泛研究的第一型 (fim)和第三型(mrk)線毛之外,其餘七套的功能以及特性都未曾被報導。我 們分別將它們命名為kpa、kpb、kpc、kpd、kpe、kpf 和 kpg。為了了解各套線毛 在特定條件下的表現情形,我們建構啟動子的活性分析系統。在各種測試條件 下,第一型以及第三型線毛的啟動子活性都比其他七套要高。此外,這兩套線毛 的活性在添加葡萄糖或甘油的培養環境下皆有顯著上升的情形。相對地,在高滲 透壓力、過氧化物的存在,或偏鹼環境的刺激下,都造成活性的降低。 另一方面,我們在 kpd 基因組上游找到一個未知功能的基因,經由胺基酸 序列比對(BLAST),我們認為其轉譯蛋白可能以雙分子訊息傳遞系統中調控子 的角色來調控kpd 基因組表現,因而命名為 kpdR。有趣的是,在克雷白氏肺炎 桿菌中大量表現 KpdR 蛋白時,我們發現 β−lactamase TEM-116 蛋白明顯增加因 而大大提高細菌對ampicillin 的抵抗力;相對的,其生物膜的形成受到抑制。雖 然,我們分別在CG43S3 和 NTUH-K2044 兩株菌中可利用核酸增殖反應(PCR)

得到 bla TEM-116,但經由比對現有的NTUH-K2044 基因資料庫卻找不到該基因。

今後,將確認 bla TEM-116的位置,並建構 kpdR 及 bla SHV-1a的突變菌株,進一步 了解 KpdR 與細菌對抗β-內酰胺類抗生素能力的關係。同時,我們在 kpb 及

(11)

kpf 基因組上游的位置分別找到類似的調控子基因,並分別命名為 kpbR 和 kpfR。而在克雷白氏肺炎桿菌中分別大量表現 KpbR 和 KpfR 蛋白時,我們發現 細菌的生長都會受到抑制。 最後,我們發現移除座落於 mrk 及 fim 基因組之間的調控基因 phgS 與 phgM,不僅會顯著降低mrk 啟動子的活性,kpg 線毛的啟動子活性也明顯下降; 而進一步,分別在這兩個啟動子Pkpg和Pmrk片段中移除過去報導我們預測 PhgS 的辨認序列,原先受PhgS 影響的現象已不復見,這些結果顯示 PhgS 和 PhgM 在 調控kpg 與 mrk 線毛的表現上扮演特定的角色。

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Abstract

There are nine fimbiral operons identified in the genome of Klebsiella

pneumoniae NTUH-K2044, a highly invasive strain isolated from Taiwan university hospital, by the HMMER search. The fim and mrk fimbriae had been described

previously, but the others are novel fimbrial operons and are respectively named kpa,

kpb, kpc, kpd, kpe, kpf, and kpg. The putative promoters of the nine operons were isolated, cloned in a LacZ reporter plasmid and the activities measured. In K.

pneumoniae CG43S3Z01, the promoters of type 1 fimbriae (PfimA and PfimB) and type 3

fimbriae (PmrkA) had higher level of activity than those of the other fimbrial promoters.

The activity of PfimA, PfimB, and PmrkA were enhanced under static cultures and the

cultures addition with glucose or glycerol. On the other hand, the expression of fimB

and mrkA were suppressed while the bacteria subject to higher osmotic pressure (>

200 mM NaCl), oxidative stress (60 μM H2O2), or the pH switch from 5.5 to 8.

Interestingly, the overexpression of KpdR, a putative response regulator located

upstream of the kpd gene cluster, appeared to decrease the biofilm formation and

increase expression of β-lactamase TEM-116 in both K. pneumoniae NTUH-K 2044

and CG43S3Z01. This implied a regulatory role of KpdR in the biofilm formation and

also the resistance to the β-lactam drug. Searching for the gene of blaTEM-116 and

(13)

over-expressions of two other putative regulators, KpbR and KpfR, in K. pneumoniae

appeared to interfere growth of the bacteria.

Finally, the deletion of phgS or phgM was found to reduce not only the

expression of type 3 fimbriae but also the activity of PkpgA. Truncation of the predicted

consensus sequences for PhgS binding from PkpgA or PmrkA abolished the PhgS

dependent expression. It was suggested that a regulatory role of PhgS for the

(14)

Introduction

The emergence of Klebsiella pneumoniae is a striking topic worldwide

Klebsiella pneumoniae, a gram-negative opportunistic pathogen, is a common cause of community-acquired and nosocomial infections (1). Although the incidence

of community-acquired K. pneumoniae has decreased, the mortality rate due to

Klebsiella pneumonia remains high (2). In western countries, most K. pneumoniae infections occur in lungs and urinary tract. While K. pneumoniae has been the leading

cause of liver abscess in Taiwan (3). It has been shown that patients with diabetic

mellitus in Taiwan are more susceptible to K. pneumoniae infection than those

without diabetes (3). In addition to liver abscess that involves destructive clinical

syndromes, the commonly emerging infections include metastatic meningitis and

endophthalmitis, osteomyelitis, and brain abscess (3, 4). Although several virulence

factors, such as polysaccharide capsule, different adhesins, lipopolysaccharide, and

iron-scavenging proteins have been identified (1), the pathogenicity of K. pneumoniae

has not been completely elucidated.

Individuals most at risk for K. pneumoniae infections are infants, the elderly, and

those with compromised defense mechanisms. Patients that suffer from Klebsiella

(15)

gastrointestinal tract by antibiotic-resistant K. pneumoniae isolates (5, 6). These

resistant strains produce extended-spectrum beta-lactamases and are of particular

concern as the infections lead to relatively high mortality rates due to treatment failure

and subsequent septicemia (7).

β-lactamases genes are prevalent in Klebsiella pneumoniae

Many Gram-negative bacteria produce β-lactamases for the resistance to

β-lactam antibiotics. The enzymes evolved from bacterial penicillin-binding proteins

which involved in peptidoglycan synthesis. The encoding genes are found on

chromosome or on transmissible plasmids (8). Plasmid-encoded β-lactamases are

often expressed in large amounts (9), while chromosomally encoded β-lactamases are

typically expressed at low levels until induced by the presence of the substrates. In

Gram-negative bacteria, β-lactamases are exported to the periplasm, whereas in

Gram-positive bacteria they are secreted from the cell (10). β-lactamases enzymes

exhibit diversity in both structure and function, and are divided into four classes (A-D)

based on their primary sequence and catalytic mechanism. Class A is the most

numerous and is a serine hydrolase as the Class C and D enzymes. The class B are

zinc-dependent β-lactamases. Historically, these enzymes were described as penicillinases because they were able to catalyze penicillin hydrolysis. The ubiquitous

(16)

belongs to class A, and was the first β-lactamase for which the crystal structure was

solved (11). Class A β-lactamases have a molecular mass of approximately 29 kDa

and are comprised of 260-280 residues, of which nine appear to be highly conserved:

four residues, Ser-70, Lys-73, Ser-130 and Glu-166 are critical for catalysis whereas

the five remaining residues Gly-45, Pro-107, Asp-131, Ala-134 and Gly-236 likely

play a role in structural integrity (12). Furthermore, three additional residues: Asn (in

most sequences)-132, Lys/Arg-234 and Ser/Thr-235 also play important roles in

enzyme activity. Sequence analysis revealed similarity between class A β-lactamases

and the low molecular weight PBPs such as PBP4 of Escherichia coli (13). In

addition, the crystal structure of class A β-lactamases revealed homology with the

catalytic domain of PBP5 from E. coli (14). However, even though the PBPs and

β-lactamases share strong structural homologies and have a common ancestry,

β-lactamases are thought to have lost the ability to interact with PG while function

efficiently as resistance enzymes (15).

The first plasmid-mediated β-lactamase in gram-negative bacteria, tem-1, was

described in the early 1960s. Another common plasmid-mediated β-lactamase found

in K. pneumoniae and E. coli is shv-1. Although SHV-1 was originally characterized

as a plasmid-mediated β-lactamase (16), recent data showing that many clinical K.

(17)

suggest its production may be intrinsic to K. pneumoniae,. Many new β-lactam

antibiotics have been designed to resist to the hydrolytic action. Resistance to these

new beta-lactam antibiotics due to extended-spectrum beta-lactamases (ESBLs) also

have emerged subsequently. ESBLs were commonly derived from tem-1 and shv-1

β-lactamases by mutations to alter the hydrolytic abilities and spectrums (17). Over

100 tem and shv types of β-lactamases have been characterized

(http://www.lahey.org/studies/webt.asp).

Biofilm formation is linked to virulence and colonization

In addition to the prevalence of ESBLs, recent studies suggest that biofilm

formation may also be an important virulence factor for K. pneumoniae. Biofilms are

organized communities of bacteria living on surface environments. Growth as a

biofilm is linked to virulence and colonization for a variety of bacterial pathogens,

including Pseudomonas aeruginosa, which causes chronic, life threatening respiratory

infections in cystic fibrosis patients (18, 19). It has been demonstrated in vitro that

bacteria growing within biofilms are more resistant to antibiotic treatment than

bacteria growing planktonically (20). While expression of β-lactamases have been

shown to inhibit bacterial biofilm formation and this anti-biofilm effect was specific

to class A and D β-lactamases (21). A model was proposed in which β-lactamases

(18)

macromolecular complexes participating in surface attachment. The interference leads

to affect the subsequent biofilm development (21).

Microbial adhesion to surfaces is the onset of the development of a biofilm.

Adhesion to host tissue receptors is the first step in successful colonization by many

Gram-negative pathogens. Adhesion fimbriae are specialized surface structures

responsible for the successful recognition and binding of these bacteria to their host

receptors. In addition, these fimbriae are responsible for maintaining this contact

during the first stages of bacterial colonization. The resulting specific adherence may

enhance bacterial persistence at the site of infections , but adherence also has been

identified as a virulence factor that facilitates tissue attack, invasion, and mucosal

inflammation.

Fimbriae are long filamentous polymeric protein structures located at the surface

of bacterial cells. The word “fimbriae” comes from the Latin word for “thread” or

“fibre” (22). These adhesive organelles are also referred to as “pili”, which comes

from the Latin word for “hair” or “hairlike strucutre” (23). Practically all

Gram-negative bacterial species that have been examined so far were found to

produce one or more types of fimbriae. While a few Gram-positive bacteria were also

(19)

proteins, and genes encoding a major subunit, minor subunit, a periplasmic chaperone,

a relatively large outer membrane usher and an adhesin protein (25). Despite their

functional differences, the conserved genetic organization implicates that these

different fimbriae evolved from a common ancestor (25).

Besides fimbriae, amorphous adhesins also exist, called a-fimbrial adhesins

(AFA) or non-fimbrial adhesins (NFA). These adhesins are present at the outer

membrane of the bacterial cell as single proteins or large multi-unit aggregates (25).

Another distinct group of adhesins is type 4 pili, which is present in a wide variety of

Gram-negative bacterial pathogens on one pole of the bacterial cell. They are, besides

adherence, associated with twitching motility (26, 27).

Polysaccharide capsule is a major pathogenicity factor

K. pneumoniae is enveloped by a prominent polysaccharide capsule that produce large, sticky colonies when plated on an agar plate with nutrient media. The strains

with the hypermucoviscosity phenotype demonstrate extremely high viscosity

determined by a string test (28). In Taiwan, the hypermucoviscosity-positive strains

were more prevalent for cases of K. pneumoniae liver abscess than from other sites of

K. pneumoniae infection (29).

(20)

with certain infection sites (30). In animal models, K1 and K2 strains are the most

virulent (1). The seroepidemiologic surveys indicated that Klebsiella K1 was rare

among North American and European clinical isolates (30, 31, 32). However, the K1

serotype was found the most frequently in Taiwan (33).

Genome-wide study of the putative chaperone-usher assembly fimbriae in K.

pneumoniae NTUH-K2044

In this study, two invasive strains of K. pneumoniae are studied and the

properties compared. K. pneumoniae NTUH-K2044 of K1 antigen, isolated from the

blood of a previously healthy 40 years-old man suffering from community-acquired

primary liver abscess and metastatic meningitis, is a hypermucoviscous strain (28). K.

pneumoniae CG43, a K2 serotype, is also a liver abscess isolate (34).

The sequence analysis revealed nine fimbrial operons identified by the HMMER

search in the genome of K. pneumoniae NTUH-K2044. Except mrk (type 3 fimbriae)

and fim (type 1 fimbriae), the other novel fimbrial operons were respectively named

kpa, kpb, kpc, kpd, kpe, kpf, and kpg. Upstream of kpb, kpd and kpf gene clusters, respectively, a gene encoding putative response regulator that contains both receiver

domain and DNA-binding domain was identified and named kpbR, kpdR, and kpfR.

(21)

namely phgS and phgM.

PecS/M plays a critical role in virulence regulation in Erwinia chrysanthemi

PecS was originally discovered to negatively regulate the expression of cellulose

and pectate lyase in E. chrysanthemi (35). The pecS mutant showed an increased rate

of infection, which is likely resulted of the overproduction of extracellular macerating

enzymes and resistance to oxidative stress (36). In addition to pectate lyase, PecS has

also been shown to negatively affect the expression of cellulase, indigoidine, a type

Ⅱsecretion pathway and flagella biosynthesis (35, 37, 38). Pectate lyases and

cellulases are extracellular enzymes that attack components of the plant cell wall and

are crucial in the pathogenesis of E. chrysanthemi (39). Indigoidine is a blue pigment

upregulated in pecS mutants and is involved in the resistance to oxidative stress (35,

36). Moreover, PecS could positively regulate the expression of polygalacturonase (40,

41).

PecS was classified into the MarR/SlyA family of transcriptional regulators.

MarR was the first member crystallized in the family (42). Although members of the

MarR/SlyA family show little sequence homology, their structures share significant

similarities. They include a common triangular shape with winged helix-turn-helix

(22)

regulatory molecules (39).

Cross-talk regulation between the expression of kpg and mrk genes

The factors mediating transcriptional regulation of fimbriae expression in

response to environmental signals either belong to the family of “local” regulators or

to the family of “global” regulators (43). The local regulators are encoded by the

respective fimbrial operons and their function is restricted to the operon by which

they are encoded. Global regulators control the expression of a variety of operons.

The regulatory activity of global regulators is superimposed over the local control of

each individual operon (44).

Global regulators which are known to be involved in fimbriae expression are,

e.g., leucine-responsive regulatory protein (Lrp), integration host factor (IHF),

catabolite gene activator protein (CAP), the histone-like protein (H-NS), and

deoxyadenosine methylase (Dam) (43). Lrp, IHF and CAP are DNA bending proteins.

The H-NS protein changes the degree of supercoiling of the DNA. Dam is an enzyme

involved in the methylation of GATC sites (43). Many fimbiral operons were found to

encode one or two local regulators. These genes probably act as transcriptional

activators of the respective operons, but their precise mode of action is not well

(23)

Specific aims

This study could be classified into three parts. In part Ι, the optimal condition for

the expression of the fimbriae is investigated using LacZ reporter system. Except for

fim and mrk gene clusters that have been well characterized in K. pneumoniae, the conditions for the optimal expression of the fimbrial operons are mysterious.

As reported by Clegg, et al. in 2006 (45), the transposon insertion in the 5’

non-coding sequence of kpg gene cluster lowered the expression level of type 3

fimbriae indicating a possible interacting regulation. It has been shown in our

laboratory that the transcription of mrk is PhgS/M dependent (46). Thus, the part ΙΙ is

to investigate the role of PhgS in regulation of kpg and mrk expression.

In the vicinity to kpb, kpd and kpf fimbrial gene clusters, three two component

system (2CS) response regulator encoding genes namely kpbR, kpdR, and kpfR were

identified. Whether the response regulator(s) play a role on the expression of the

(24)

Materials and Methods

Bacterial strains, plasmids and growth conditions

Bacterial strains and plasmids used in this study are listed in Table 1. E. coli

JM109 was used for cloning and Pseudomonas aeruginosa PAO1 was a positive

control strain in biofilm formation assay. Bacteria were grown at 37 ℃ in

Luria-Bertani (LB) broth or the medium supplemented with appropriate antibiotics.

Antibiotics were used where indicated at the following concentrations: ampicillin

(100 μg/ml), kanamycin (25 μg/ml), and chloramphenicol (35 μg/ml). For

β-galactosidase activity assay, the bacteria in different culture conditions were

statically grown to early logarithmic phase (optical density at 600 nm 0.4 to 0.5).

DNA manipulation

Plasmids were purified by the High-Speed Plasmid Mini kit (Geneaid, Taipei,

Taiwan). All restriction and DNA-modifying enzymes were used as recommended by

the manufacturer (Fermentas, Hanover, MD, USA). PCR amplifications were

performed with Taq DNA polymerase (MDBio, Inc, Taiwan) or Blend TaqTM-Plus-

DNA polymerase (Cosmo Bio Co., LTD.). PCR products and DNA fragments were

purified using the Gel/PCR DNA Fragments Extraction kit (Geneaid). The primers

(25)

cells was performed by following the method of Dower (47).

Construction of plasmid-mediated LacZ reporter strains in K. pneumoniae CG43S3Z01

The putative promoter containing about 600 nucleotides upstream of the

translation initiation codon of each of the fimbrial gene clusters (Fig. 1) were PCR

amplified with respective primer pairs (Table 2) from the genomic DNA of K.

pneumoniae NTUH-K0244, and the PCR product cloned into BamHI site in front of the promolerless lacZ in the low-copied plasmid, pLacZ15 (48). To identifiy the

Phg-binding site in PkpgA and PmrkA, the specific primers ZY105/pCC046,

ZY106/pCC046 and ZY107/pMrkA5 were used to generate the truncated forms of the

promoters.

Measurement of β-galactosidase activity

Miller assay was carried out as described (48). In summary, a culture was grown

under the tested condition to early logarithmic phase (optical density at 600 nm 0.4 to

0.5). A 100 μl was removed and added to the reaction mixture which containing 900

μl Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM

β-mercaptoethanol), 17 μl 0.1% SDS and 35 μl chloroform. The reaction mixture was

(26)

ONPG was added to the reaction and incubated at 30℃ until yellow color was

apparent or for 1 h if there was no obvious color change. Finally, the reaction was

stopped by adding 500 μl of stop solution (1 M Na2CO3) and the absorbance of the

supernatant was measured at OD420 (ELx800, BIO-TEK). One unit of β-galactosidase

is defined as the hydrolysis of 1 nmol ONPG per min per mg protein. Strains were

tested under different conditions of temperature, pH, oxygen levels, carbon source,

serums, osmolarity stress, and others.

Over-expression of KpbR, KpdR and KpfR

The kpbR, kpdR and kpfR were respectively amplified with specific primers

(Table 2) and cloned into a TA vector, pCR®

2.1-TOPO®

(Table 1), and then subcloned

into pETQ33, which carrying an IPTG-inducible tac-promoter preceding the multiple

cloning site, with EcoRI digestion. The resulted recombinant plasmids were then

transformed into E. coli JM109, K. pneumoniae NTUH-K2044 and K. pneumoniae

CG43S3 strains. Analysis of the overexpression was carried out while these

transformants were induced with 0.5 mM IPTG and cultured at 37℃ for 4 h.

In-gel digestion

Stained protein bands were excised from gels and were rinsed with distilled

(27)

bicarbonate and acetonitrile (1:1) for 15 min, and then the gels were transferred into

another solution containing 50 mM ammonium bicarbonate and acetonitrile (3:2) for

15 min. After repeating the destain procedure, the protein was incubated in 500 μl

acetonitrile to remove the water and dried using a speed vaccum concentrator. After

immersed in 50 μl of 25 mM ammonium bicarbonate, the protein was digested with 3

to 5 μl trypsin (20 μg/ml) at 37℃ for 12 to 16 h. The digested peptides were then

recovered using a solution containing 0.1% trifluoroacetic acid (TFA) in acetonitrile.

The resulting peptide extracts were processed to MS or MS/MS analysis by core

facilities for proteomics and structural biology research in Academia Sinica

(http://proteome.sinica.edu.tw/).

Ampicillin susceptibility assay

Ampicillin susceptibility test was performed by the broth method (49). An

overnight culture of bacteria was refreshed in LB broth with different concentrations

of ampicillin. The transformants of K. pneumoniae NTUH-K2044 and K. pneumoniae

CG43S3 carrying pETQ33 or kpdR overexpression plasmid were cultured with

addition of 0.5 mM IPTG and 25μg/ml kanamycin. The optical density at 600 nm

were measured after 16 h of incubation at 37℃.

(28)

Biofilm formation was assessed by the ability of the cells to adhere to the glass

tubes. The indicator medium contained an aliquot of 1:20 diluted overnight bacteria

culture in LB and was incubated at 37 ℃ for 48 h for biofilm formation.

Quantification of biofilm formation was carried out as the reported protocol (50) with

some modification. Essentially, the indicator medium (200 μl/well) in 96-well

microtire dishes made of PVC (TPP 96 flat) contained an aliquot of 1:10 diluted

overnight grown bacteria in LB and the plate was incubated at 37℃ for 12 h, 24 h or

48 h. The unadherent bacteria was washed with 200 μl H20 and then 200 μl of 1%

crystal violet was added to each well. After the plate was placed at room temperature

for 30 min, ddH2O was used to wash the plate three times. Finally, the crystal

violet-stained biofilm was solubilized in 200 μl of 0.1% SDS and the absorbance

determined at OD595 using spectrophotometer.

Yeast agglutination assay

The capacity of bacteria to express a D-mannose-binding phenotype was

determined by their activity to agglutinate yeastcells (Saccharomyces cerevisiae) on

glass slide. Bacterial cells were adjusted to about 1X109 CFU and mixed with yeast

cells (0.01%, in PBS) until the time agglutination occurred was observed in different

(29)

Qualitative analysis of the capsule polysaccharide (CPS)

Qualitative analysis of the CPS was performed by sedimentation test. An

overnight culture of bacteria was refreshed in LB broth with 0.5 mM IPTG and

25μg/ml kanamycin. The sedimentation test was carried after 8 h of incubation at

37℃ and centrifuged at 3000 rpm for 10 min.

Preparation of anti-KpdA, anti-KpfA and anti-KpgA polyclonal antibodies

Five-week-old female BALB/c mice, purchased from Laboratory Animal Center

in National Taiwan University, received injection in peritoneal cavity with about 50

μg/150 μl gel-extracted KpdA, KpfA or KpgA proteins respectively on day 1, 11 and

15. For the first injection, the same volume of Freund’s complete adjuvant was used,

and Freund’s incomplete adjuvant was used for the following injections. The KpdA,

KpfA and KpgA antisera were finally obtained by intracardic puncture. Indirect

ELISA was performed to test the titer of serum from the tail on day 15 and the serum

was harvested from the blood of the mice’s heart.

Western blot analysis

As the method described (51), total cell lysates were resolved by 12.5% sodium

dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins

(30)

(ImmobilonTM-P, Millipore). Subsequently, the membranes were blocked with 5%

skim milk in PBS, the membranes were incubated with a 10,000-fold diluted KpdR,

KpfR, KpgR or MrkA anti-serum at room temperature for 2 h. Followed by

incubation with a 10,000-fold diluted alkaline phosphatase-conjugated anti-mouse or

anti-rabbit (to MrkA) immunoglobulin G at room temperature for 1 h, additional

washes were applied triply and the bound antibodies were detected by using the

chromogenic reagents BCIP (5-bromo-4-chloro-3-indolyl phosphate) and NBT (Nitro

blue tetrazolium).

Localization of the blaTEM-116 in K. pneumoniae NTUH-K2044

Extracted genomic DNA was treated with NotI, BamHI, and EcoRV digestions

and the fragmented DNA cloned to NotI, NotI/BamHI, BamHI/EcoRV, NotI/EcoRV,

EcoRV, and BamHI sites of pET30a individually. Then these clones in E. coli JM109 were selected with ampicillin and kanamycin.

Extraction of mRNAs from K. pneumoniae

Total RNA was isolated from mid-exponential phase of K. pneumoniae CG43S3

or NTUH-K2044 cells (OD600=0.6~0.8) by extraction with the TRI reagent

(Molecular Research Center, Cincinnati, OH). Contaminating DNA was eliminated

(31)

Total RNA (1 μg) was reverse-transcribed using SMART PCR cDNA synthesis kit

(32)

Results

Part Ⅰ

(33)

Comparative analysis of the fimbrial gene clusters present in K. pneumoniae CG43S3

In the sequenced genome of K. pneumoniae CG43S3 (http://genome.nhri.org.tw

/kp/), we could find seven other fimbrial gene clusters, except kpb and kpc. An

incomplete kpb gene cluster was found in K. pneumoniae CG43S3. The KpbD

encoding gene, which encodes a putative adhsin of kpb fimbriae, could not be

identified by PCR analysis in K. pneumoniae CG43S3. However, kpbB gene encoding

a putative chaperone could be found.

In vitro expression of the fimbrial genes under different conditions

Bacteria only fully express virulence factors when the conditions are appropriate

within a host. Therefore, bacteria must have a way of recognizing the environmental

circumstances and be able to adapt their protein expression according to these

conditions. Regulation of fimbiral gene expression triggered by environmental signals

in enteric pathogens is well documented. The environmental conditions include

temperature, carbon source, osmolarity, pH, and so on (43).

The promoter regions of each of the putative fimbrial gene clusters (except kpc

and kpe) were cloned to the lacZ reporter plasmid pLacZ15 and the β-galactosidase

(34)

majority of the fusion promoters exerted extremely low level of activity. Only the

putative promoter of kpaA, fimA, fimB and mrkA showed a certain level of detectable

activity. A slightly higher expression was noted when the strains were grown in LB

than in M9 medium (data not shown). As shown in Table 3, with exception of the PfimE

activity, all the others appeared to be higher in standing culture than in the aerated

condition. As the temperature decreased from 37℃ to 30℃, the expression of kpaA,

kpfA, kpgA, fimA, fimB and mrkA in standing culture increased. The promoter activity of fimB and mrkA at 30℃ were found to be about three times higher than those at 37

℃. While the growth at mild acidic condition (pH 5.5), apparent increase of the

expression of fimB and mrkA were also observed. Moreover, addition of 0.2%

glycerol or 10% bovine albumin serums exerted a positive effect on the promoter

activity of fimB and mrkA. Notably, the increased expression of fimA expression was

significant with the addition of 10% bovine albumin serums (Table 4). However, as

the growth at the condition adding with different concentrations of sodium chloride,

H2O2 or bile salts treatments had negative effect on the expression of fimB and mrkA

(Table 5).

The phgS deletion effect on the fimbrial expression

(35)

on each of the promoter activity was hence investigated. As shown in Fig. 2, the

activity of PmrkA was drastically decreased. Interestingly, PkpgA was also found to be

reduced greatly in the phgS deletion mutant K. pneumoniae CG43S3Z01phgS-.

The possible interacting regulation for the expression of kpg and mrk in K.

pneumoniae 43816 has been previously implicated by Clegg, et al (45). They reported an insertion in the upstream non-coding sequence of kpg fimbrial gene cluster

decreased the expression of type 3 fimbriae (mrk) (45). We speculate that PhgS is

likely the regulator for the interacting regulation between kpg and mrk.

Since phgS/M are homologous genes of pecS/M of E. chrysanthemi. PecS,

belonging to the MarR family controls the synthesis of various virulence factors (39).

The crystal structure of MarR had been recently elucidated (42). Sequence alignment

with the representative members of the MarR family indicated that PhgS shares high

similarity in the DNA-binding domain (Fig. 3). A consensus binding site for MarR

family was therefore predicted. The analysis of 11 PecS target genes revealed a

consensus binding site which further demonstrated by mutation analysis (38). The

binding element consists of a 23-bp palindrome-like sequence

(C-11G-10A-9N-8W-7T-6C-5G-4T-3A-2)T-1A0T1(T2A3C4G5A6N7N8N9C10G11). The very

conserved part -6 to 6 allows a specific interaction with PecS and the relatively

(36)

four bases G-4, A-2, T2 and C4 are required for efficient binding of PecS and the

presence of several binding sites on a promoter increases its affinity to PecS (38).

Comparative analysis with the conserved sequence, a putative palindrome with

the conserved nucleotides were found to be present on PkpcI, PkpgA, and PmrkA. Two

putative PhgS-binding sites were found on PkpgA (Fig. 4). As shown in Fig. 5, a

putative PhgS binding site is located within PmrkA which is about 380 bp upstream of

the mrkA start codon. In order to verify if the consensus sequence is required for the

PhgS dependent expression, truncation forms of Pkpg and PmrkA including PkpgA1 (420

bp), PkpgA2 (488 bp), and PmrkAt (386 bp) were generated. As shown in Fig. 6, the

deletion of phgS appeared to be no effect on the promoter activity of either PkpgA1

PkpgA2, or PmrkAt implying that the truncated sequence is a PhgS binding element.

(37)

Part Ⅱ

Characterization of the putative regulators encoding by the genes

respectively located in the vicinity to kpb, kpd, or kpf gene cluster

(38)

Characterization of the putative response regulator KpbR

The locus kpbR is located 451 bp upstream of the kpb gene cluster in a divergent

transcription orientation. As shown in Fig. 7, next to kpbR, there is a putative

entericidin locus in both KP strains. E. coli entericidin has been implicated to be

required for the bacterial programmed cell death (52). Downstream of the entericidin

loci ecnA and ecnB, a 500 bp DNA encoding elongation factor P was found (52). In

Citrobacter freundii. ecnR encoding a member of the OmpR response regulator family was found upstream of ecnB. Next to ecnB, sugE gene which has been reported to

encode a member of the SMR family of small multidrug resistance efflux pump (53)

could be identified in the three bacteria (Fig. 7). Sequence analysis revealed that

KpbR encodes a 2CS response regulator with an OmpR domain. The response

regulator of approximately 25.1 kDa contains a carboxy-terminal DNA-binding

helix-turn-helix motif and a conserved aspartate residue near the amino terminus (Fig.

8).

While overexpress KpbR in E. coli JM109, an apparent synthesis of a protein

with the predicted molecular weight of KpbR could be observed (Fig. 9). However,

transformation of pKpbR into NTUH-K2044 or CG43S3 appeared to inhibit growth

(39)

Characterization of the putative response regulator KpdR

As shown in Fig. 10A, kpdR is located upstream of kpd gene cluster in a

divergent transcription orientation with an intergenic sequence of 414 bp. The Blast

analysis revealed that KpdR is a putative response regulator with a carboxyl

DNA-binding motif and a conserved aspartate at the N-terminal CheY-like receiver

domain. Notably, the over-expression of the KpdR in both K. pneumoniae strains

enhanced the synthesis of a protein of about 28.5 kDa (Fig. 10B). The protein band

was then isolated, subjected to in-gel digestion by trypsin and MS/MS analysis for

protein identity. As shown in Fig. 10C, the specific peptide

QQLIDWMEADKVAGPLLR matched to the sequence of β-lactamase TEM-116.

Nevertheless, we found no sequence homolog of TEM-116 gene in either genome of

K. pneumoniae NTUH-K2044 or CG43 (http://genome.nhri.org.tw/kp/). In order to confirm the protein identity, two pairs of TEM116 specific primers (56) were

synthesized for amplification of L1 and L2 products as shown in Fig. 11A. As shown

in Fig. 11B, the TEM-116 specific PCR products could be detected in K. pneumoniae

NTUH-K2044 and CG43 strains indicating the TEM116 gene is present in both

strains.

To investigate if the increased synthesis of TEM-116 affects the bacterial

(40)

shown in Fig. 12, the KpdR over-expression appeared to drastically increase the

bacterial resistance to ampicillin. This further supported that over-expression of KpdR

enhances the expression of TEM-116 β-lactamase.

While overexpression of FimB in K. pneumoniae CG43 (Fig. 13A), apparently

diminished expression of MrkA, the major pilin of type 3 fimbriae was observed (Fig.

13B). However, the overexpression of KpdR had no apparent effect on the pilin

synthesis as determined by western blot hybridization in Fig. 13. In K. pneumoniae,

MrkA pilin of type 3 fimbriae has been shown as the major determinant for the

bacterial activity to form biofilm (45). In case that the effect of the KpdR

overexpression on type 3 fimbrial expression is too subtle to be detected, the effect on

the biofilm formation was analyzed. The overexpression of KpdR reduced the biofilm

formation when compared to that of wild-type bacteria carrying vector plasmid (Fig.

14). However, no apparent changes could be observed when the biofilm formation

capability was quantitatively determined using 96-well PVC-microtire dish (data not

shown). Moreover, the over-expression of KpdR slightly decreased the synthesis of

the capsular polysaccharide (Fig. 15).

Characterization of the putative response regulator KpfR

(41)

and a CheY-like receiver domain containing the conserved aspartate residue at the

amino terminus (Fig. 16). The entire coding region of kpfR has been isolated and

cloned into expression vector pETQ30a. However, the optimal condition for the

(42)

Discussion

Fimbriae are a focus for research as they often define the initial interaction with

the host and are a key target for development of interventions such as vaccines. The

investigation into the expression of these fimbriae is contributive to image of the

disease dynamics during infection. We have found in this work that transcription of

the fimbrial operons, except kpd, were enhanced by growing at 30℃. Temperature has

been shown to be an important regulator of virulence gene expression in several

genera of bacteria. Cytoplasmic membranes, nucleic acids, and ribosomes are

suggested to be the cellular thermosensors (54). The major effects of lower

temperature are a decrease in membrane fluidity and the stabilization of secondary

structures of RNA and DNA, which may affect the efficiency of translation,

transcription, and DNA replication. Specific transcription factors may involve in the

expressions of these putative fimbriae at lower temperature.

As shown in Table 4, higher levels of promoter activity were observed for both

PfimB and PmrkA as the bacteria were treated with glucose, glycerol, or serum. The mild

acidic environment also appeared to enhance both PfimB and PmrkA activities. Some

pH-responsive regulatory mechanisms involved alternative σ factors that sense an

acidifying environment (54). However, whether the expression of fim or mrk involves

(43)

intestine, a natural habitat of K. pneumoniae, where they act as detergents for the

digestion of fats. In humans, 96% to 99% of the bile salts entering the small intestine

are absorbed and returned to the liver, and only 1% to 4% pass the ileocecal valve and

enter the colon (55). K. pneumoniae could have evolved adaptive strategy for dealing

with bile salts. Nevertheless, no apparent effect of bile salts on the fimbrial expression

was found.

Since there are multiple fimbriae in K. pneumoniae, investigation of the

regulatory network in fimbrial expression is ponderable. Both K. pneumoniae

NTUH-K2044 and CG43S3 appeared to express MrkA protein in LB static culture at

37℃ (Fig. 13). The activity of PmrkA has been shown to be dropped greatly as phgS or

phgM was deleted from K. pneumoniae CG43S3Z01 (46). As shown in Fig. 2, deletion of phgS or phgM was also found to lower the activity of PkpgA (Fig. 2). A

possibly interacting regulation between kpg and mrk has been discussed in K.

pneumoniae 43816 (45). We demonstrated here that PhgS/M likely play, a role on the interacting regulation.

The entericidin locus has been implicated in determining programmed cell death

(PCD) in bacteria (52). One of the best-studied PCD in bacteria is called

toxin-antitoxin system, which consists of a pair of genes that specify two components:

(44)

toxin. In 1998, a novel chromosomal bacteriolytic module called entericidin locus was

reported in E. coli and C. freundii (52). The entericidin locus contains two genes,

ecnA and ecnB, which appear to encode two small, amphipathic α-helical lipoproteins. That putative antidote/toxin pair is positively controlled by RpoS and negatively

controlled by the osmosensing EnvZ/OmpR. Under high osmolarity in stationary

phase, the locus promotes bacteriolysis with the ecnA gene product acting as an

antidote to EcnB (52). Since, we could not find ecnA next to ecnB gene in either K.

pneumoniae NTUH-K2044 or CG43S3 genome, the involvement of the locus in regulation of KpbR is elusive.

In Korea, the blaTEM-116 has been identified recently (56, 61). The prevalence

study of TEM-type β-lactamase-producing K. pneumoniae indicated that blaTEM-116 is

the predominant type in Taiwan and Guangzhou (56, 57). Why the over-expression

of KpdR enhances the β-lactamase TEM-116 expression is considerably urgent and

important. The over-expression of KpdR also affected biofilm formation and capsular

synthesis, which supporting the mechanism that was proposed the β-lactamase

interferes with peptidoglycan remodeling complexes participating in surface

(45)

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Klebsiella pneumoniae CG43 in a coordinated manner. J. Biochem. (Tokyo). 2006; 140: 429-38.

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62. Tzouvelekis LS, Bonomo RA. SHV-type beta-lactamase. Curr. Pharm. Des.

(55)

Table 1. Bacterial strains and plasmids used in this study (A)

Strain or plasmid Description Reference

Strain K. pneumoniae CG43S3 CG43S3 Smr (58) CG43S3-Z01 CG43S3 ΔlacZ (60) CG43S3mrkA- CG43S3-Z01 ΔmrkA (46) CG43S3phgS- CG43S3-Z01 ΔphgS (46) CG43S3phgM- CG43S3-Z01 ΔphgM (46) E. coli

JM109 RecA supE44 endA1 hsdR gyrA96 relA1 thi (lac proAB)F [lacIq lacZ M15 proAB traD36] Our Lab.

S17-1λpir HsdR recA pro RP4-2 [Tc::Mu; Km::Tn7] (λpir) (59)

P. aeruginosa

PAO1 Wild-type strain, laboratory strain serogroup 05 Our Lab.

Plasmid

Yt&A vector PCR cloning vector, Apr Sigma

Pcr®

2.1-TOPO®

PCR cloning vector, Kmr Invitrogen

pLacZ15 A derivative of Pyc016, containing a promoterless lacZ from K. pneumoniae CG43S3 as the reporter, Cmr (60)

Petq33 Protein expression vector, Kmr Our Lab.

PPfimA ~500-bp BamHI fragment containing the putative fimA promoter, cloned into BamHI site of placZ15 (46)

pPfimB ~500-bp BamHI fragment containing the putative fimB promoter, cloned into BamHI site of placZ15 (46)

pPfimE ~500-bp BamHI fragment containing the putative fimE promoter, cloned into BamHI site of placZ15 (46)

(56)

(B)

Plasmid Description

pPkpaA 580-bp BamHI fragment containing the putative kpaA promoter, cloned into BamHI site of placZ15

pPkpbA 639-bp BamHI fragment containing the putative kpbA promoter, cloned into BamHI site of placZ15

pPkpbR 639-bp BamHI fragment containing the putative kpbR promoter, cloned into BamHI site of placZ15

pPkpdA 602-bp BamHI fragment containing the putative kpdA promoter, cloned into BamHI site of placZ15

pPkpdR 602-bp BamHI fragment containing the putative kpdR promoter, cloned into BamHI site of placZ15

pPkpfA 746-bp BamHI fragment containing the putative kpfA promoter, cloned into BamHI site of placZ15

pPkpgA 578-bp BamHI fragment containing the putative kpgA promoter, cloned into BamHI site of placZ15

pPkpgA1 420-bp BamHI fragment containing the putative kpgA promoter, cloned into BamHI site of placZ15

pPkpgA2 388-bp BamHI fragment containing the putative kpgA promoter, cloned into BamHI site of placZ15

pPmrkAt 386-bp BamHI fragment containing the putative mrkA promoter, cloned into BamHI site of placZ15

pKpdR 867-bp EcoRI fragment containing the kpdR of K. pneumoniae NTUH-K2044, cloned into pETQ33, Kmr

pKpbR 843-bp EcoRI fragment containing the kpbR of K. pneumoniae NTUH-K2044, cloned into pETQ33, Kmr

(57)

Table 2. Primers used in this study

Primer Sequence Complementary position

ZY001 5'-CAT ATGAAAAAATACAGCAGAGGAATGG-3' -3 relative to the kpdA start codon ZY002 5'-GCGGGTGGAGCATAACTTTG-3' +371 relative to the kpdD stop codon ZY003 5'-CATATGAAAATGAAATCACTTTGCCTGG-3' -3 relative to the kpfA start codon ZY004 5'-ATGGTGACTTTCGCCCTGGAG-3' +45 relative to the kpfD stop codon ZY005 5'-CATATGAAAAAACAACCTCGCTTTATAACC-3' -3 relative to the kpgA start codon ZY006 5'-CGAGAACGCCGAAGCTGG-3' +77 relative to the kpgD stop codon ZY007 5'- GATCCGAATTCGCCAAAACC -3' +1289 relative to the kpgC start codon ZY008 5'- GGTTTTGGCGAATTCGGATC -3' -1284 relative to the kpgC stop codon ZY009 5’-CTGCGGGATCCTGTGGGCTG-3’ -102 relative to the kpbR start codon ZY010 5’-GACCAGCAAATGACTATCGCACC-3’ +52 relative to the kpbR stop codon ZY011 5’-CCAGGGGATCCTTATGTTGATCTGC-3’ -13 relative to the kpdR start codon ZY012 5’-CTGGGCGTGGCGAGTAATG-3’ +177 relative to the kpdR stop codon ZY013 5’-CATGCAACATCTTTCATTAGGATCCTTC-3’ -30 relative to the kpfR start codon ZY014 5’-CCGACGAGTGCCATTGCCAG-3’ +41 relative to the kpfA start codon

ZY101 5'-CGCTCATGAGACAATAACCC-3' -56 relative to the blaTEM-116 start codon (56)

ZY102 5'-CAGTGAGGCACCTATCTC-3' -51 relative to the blaTEM-116 stop codon (56)

ZY013 5’-CATTTTGCCTTCCTGTTTTTG-3’ +67 relative to the blaTEM-116 start codon

ZY104 5’-CTACGATACGGGAGGGCTTACC-3’ -129 relative to the blaTEM-116 stop codon

ZY105 5'-GTAGGATCCGACGAGCGCAC-3' -442 relative to the kpgA start codon ZY106 5'-GAATGGATCCGTTGTTGTTTAAAGG-3' -373 relative to the kpgA start codon ZY107 5'-CTGGATCCTGTTGCGGTC-3' -358 relative to the mrkA start codon pCC033 5’-CGTGCGCTGGATCCTGTT-3’ -532 relative to the kpaA start codon pCC034 5’-TAAAAGATCTATGGCGGGTGC-3’ +50 relative to the kpaA start codon pCC035 5’-GCATTGAGGCGGATCCACT-3’ -597 relative to the kpbA start codon pCC036 5’-GGTCGCTAGATCTGCAGTGC-3’ +46 relative to the kpbA start codon pCC039 5’-CGGATCCGCATATGCTGA-3’ -555 relative to the kpdA start codon pCC040 5’-GCCGCTGACGGGAAGATCT-3’ +46 relative to the kpdA start codon pCC043 5’-ACAGGCCGGATCCATGAC-3’ -681 relative to the kpfA start codon pCC044 5’-GCTGGCCTTAGATCTGGCTAC-3’ +59 relative to the kpfA start codon pCC045 5’-GGATCCGGTCTCTGGTTAACAT-3’ -526 relative to the kpgA start codon pCC046 5’- CCATCCTCATAGGAGCGCTGCT-3’ +47 relative to the kpgA start codon

(58)

Table 3. Effect of culture condition on the fimbrial expression

Promoter activity (Miller units)a

Fimbriae

Growth conditions kpaA kpdA kpdR kpfA kpgA fimA fimB fimE mrkA

Aerated culture (37℃) 183.2 (±10.7) 21.7 (±1.9) 26.3 (±1.7) 23.0 (±2.8) 92.8 (±3.6) 520.4 (±27.3) 32.3 (±0.7) 96.1 (±5.5) 27.8 (±4.0) Standing culture (37℃) 272.6 (±12.9) 24.8 (±1.1) 30.4 (±1.4) 46.3 (±2.1) 92.8 (±5.7) 510.8 (±25.6) 806.2 (±37.7) 24.3 (±1.1) 791.6 (±26.1) Temperature 30℃ 372.7 (±8.0) 16.8 (±0.6) 25.3 (±0.7) 90.8 (±2.8) 221.6 (±13.1) 594.4 (±32.9) 2555.9 (±53.0) 25.1 (±0.8) 2505.9 (±59.9) a, the fimbrial expression was determined using LacZ as the promoter reporter. LacZ activity measurement was as described in Materials and Methods.

(59)

Table 4. Effects of glucose, glycerol, and bovine serum on the fimbrial expression

Promoter activity (Miller units)a

Fimbriae

Growth conditions kpaA kpdA kpdR kpfA kpgA fimA fimB fimE mrkA

Standing culture 272.6 (±12.9) 24.8 (±1.1) 30.4 (±1.4) 46.3 (±2.1) 92.8 (±5.7) 510.8 (±25.6) 806.2 (±37.7) 24.3 (±1.1) 791.6 (±26.1) 15 mM 244.1 (±22.2) 22.0 (±1.9) 37.8 (±0.5) 43.0 (±1.3) 93.0 (±2.6) 504.2 (±27.1) 1217.4 (±32.7) 22.6 (±1.8) 1753.2 (±38.2) Glucose 30 mM 215.1 (±11.9) 23.2 (±0.7) 33.2 (±1.1) 45.6 (±1.9) 88.3 (±2.1) 511.2 (±26.3) 1112.6 (±39.5) 20.7 (±1.1) 1563.8 (±33.3) Glycerol 0.2% 345.1 (±9.7) 23.8 (±0.7) 34.4 (±1.1) 40.7 (±1.5) 114.1 (±9.1) 512.6 (±22.4) 3266.5 (±49.2) 25.6 (±1.8) 3431.0 (±58.7) Bovine serum 10% 309.3 (±14.8) 23.0 (±1.1) 27.4 (±1.9) 51.9 (±6.0) 108.5 (±11.4) 1872.9 (±32.6) 1984.7 (±28.3) 19.9 (±1.4) 1617.2 (±27.7) a, the fimbrial expression was determined using LacZ as the promoter reporter. LacZ activity measurement was as described in Materials and Methods.

(60)

Table 5. Effects of pH, osmotic pressure, H2O2, and bile salts on the fimbrial expression

Promoter activity (Miller units)a

Fimbriae

Growth conditions kpaA kpdA kpdR kpfA kpgA fimA fimB fimE mrkA

Standing culture 272.6 (±12.9) 24.8 (±1.1) 30.4 (±1.4) 46.3 (±2.1) 92.8 (±5.7) 510.8 (±25.6) 806.2 (±37.7) 24.3 (±1.1) 791.6 (±26.1) 5.5 270.9 (±4.7) - - 26.9 (±3.2) 104.6 (±15.6) 174.0 (±17.3) 2170.2 (±88.6) 30.3 (±7.2) 2335.1 (±94.5) 8.0 195.9 (±2.8) 27.9 (±1.8) 49.5 (±6.2) 32.2 (±1.4) 82.9 (±17.5) 368.8 (±28.4) 232.8 (±10.1) 25.9 (±2.8) 292.8 (±15.6) pH 30 mM 215.1 (±11.9) 23.2 (±0.7) 33.2 (±1.1) 45.6 (±1.9) 88.3 (±2.1) 511.2 (±26.3) 1112.6 (±39.5) 20.7 (±1.1) 1563.8 (±33.3) 0 mM 129.0 (±22.4) 23.6 (±1.4) 27.0 (±1.0) 23.0 (±2.3) 56.6 (±7.3) 202.5 (±16.1) 61.8 (±3.0) 13.8 (±1.0) 57.7 (±3.8) 200 mM 120.1 (±15.6) 23.7 (±0.9) 26.8 (±1.8) 17.6 (±17.6) 59.2 (±6.0) 220.8 (±24.3) 432.5 (±22.7) 14.1 (±0.6) 381.2 (±24.3) 800 mM 106.5 (±9.2) 18.9 (±1.4) 34.2 (±1.7) 14.8 (±1.1) 51.4 (±3.3) 165.4 (±12.9) 39.3 (±2.6) 12.5 (±0.7) 47.1 (±1.3) NaCl 1.5 M 111.3 (±8.3) - - 15.2 (±2.4) 31.4 (±2.3) 154.9 (±10.1) 28.8 (±1.5) 14.3 (±2.6) 32.3 (±2.5) H2O2 60 μM 191.5 (±19.8) 22.9 (±1.5) 32.1 (±1.5) 27.4 (±2.8) 100.2 (±2.3) 247.9 (±15.1) 606.0 (±18.4) 23.3 (±1.4) 575.2 (±19.1) Bile salts 0.3% 103.0 (±10.0) 5.2 (±0.7) 8.1 (±0.2) 20.3 (±0.3) 43.1 (±3.8) 224.1 (±6.7) 553.7 (±15.0) 7.1 (±0.7) 469.7 (±17.6) a, the fimbrial expression was determined using LacZ as the promoter reporter. LacZ activity measurement was as described in Materials and

(61)

Figure 1. Gene organization of the putative fimbrial gene clusters in K. pneumoniae NTUH-K2044. The putative promoters are

respectively boxed. The KP number is according to the annotation of the genome (http://genome.nhri.org.tw/kp/). phgM phgS regulator recombinase adhesin usher chaperone pilin subunit fim [type 1] (KP4570~4579) mrk [type 3] (KP4555~4559) kpg (KP4248~4251) kpf (KP4244~4247) kpe (KP2716~2719) kpd (KP1401~1404) kpc (KP0596~0601) kpb (KP0409~0412) kpa (KP0334~0338)

(62)

Miller units

0 200 400 600 800 K. pneumoniae CG43S3Z01 K. pneumoniae CG43S3Z01 phgS deletion

PkpaA PkpdA PkpfA PkpgA PfimA PfimB PfimE PmrkA

Figure 2. Measurements of the fimbriae in K. pneumoniae CG43S3Z01 and CG43S3Z01phgS-. This assay was carried out as described in Materials and Methods while the bacteria was statically grown in LB at 37℃ to exponential growth phase.

數據

Table 1. Bacterial strains and plasmids used in this study  (A)
Table 2. Primers used in this study
Table 3.    Effect of culture condition on the fimbrial expression
Table 4.    Effects of glucose, glycerol, and bovine serum on the fimbrial expression
+7

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