Thesis objectives

The prevalent presence of 2CS encoding genes in the bacterial genome as well as the wide spectrum of physiological functions governed by different 2CSs, such as virulence properties, have aroused our interest in studying the 2CSs in K. pneumoniae. Based on the available E.

coli 2CSs listed in the KEGG database (http://www.genome.ad.jp/kegg/pathway/), a previous search of the homologous 2CS-coding genes in the genome sequence of K. pneumoniae MGH78578 (http://genome.wustl.edu/) has been performed. Among the 29 sensor kinase coding genes and 26 response regulator coding genes identified, the 2CSs PhoP/PhoQ, PmrA/PmrB, RstA/RstB and RcsCDB coding genes were also included. The role of 2CSs PhoP/PhoQ, PmrA/PmrB in S. enterica pathogenesis has been well documented (2, 13, 78, 205,

260). A novel mechanism connecting 2CSs PhoP/PhoQ and PmrA/PmrB by a small protein PmrD has shed light on the complex and versatile regulation of 2CS target genes (59, 117).

RstA/RstB has been identified as a member of magnesium stimulon governed by the master 2CS PhoP/PhoQ (164). Recent studies have shed light its functional role in bacterial acid resistance (214), iron transport (111) and the survival against stressed encountered in the digestive tract (69). Despite these findings, the function of RstA/RstB remained rather limited.

In addition, the RcsCDB phosphorelay has been reported to regulate E. coli group I CPS (87) and K. pneumoniae K2 CPS biosynthesis (245). In K. pneumoniae, in addition to RmpA2 which activated K2 cps expression independently to RcsB (130), another mucoid factor encoding gene rmpA was also indentified in pLVPK (37). Based on the critical role of CPS in K.

pneumoniae pathogenesis, it would be of importance to investigate the mode of regulation of RmpA as well as its interplay with the RcsCDB phosphorelay. The objectives of this thesis are to investigate the functional role of PhoP/PhoQ, PmrA/PmrB, RstA/RstB and RcsCDB in the regulation of K. pneumoniae virulence determinants, and an overview is listed as follows:

Chapter 2 described the functional role of bacterial CPS as well as 2CSs PhoP/PhoQ and PmrA/PmrB in the regulation of polymyxin B resistance in K. pneumoniae CG43. In addition to the characterization of the PmrD connector-mediated pathway in K. pneumoniae at both genetic and the molecular level, the residues implicated in PmrD functioning were also explored via a mutagenesis approach.

Chapter 3 presents the characterization of the 2CS RstA/RstB in K. pneumoniae CG43.

The involvement of PhoP and RstA on the expression of rstA was investigated, and RstA-regulated genes were identified by subtractive cDNA hybridization. Based on the results from cDNA subtraction and recent findings in RstA/RstB, phenotype analysis was conducted to explore the possible biological functions of RstA/RstB in K. pneumoniae.

Chapter 4 reports RmpA regulation of K. pneumoniae K2 CPS biosynthesis. The cooperation of RmpA and the RcsCDB phosphorelay was implicated from phenotype analysis and was demonstrated through both in vivo and in vitro approaches. A comparative analysis of RmpA and RmpA2 has further led to the identification of Fur, a global iron uptake regulator, in Klebsiella K2 CPS biosynthesis as well as the interplay of iron uptake and capsule biosynthesis in K. pneumoniae pathogenesis.

Chapter 5 concludes the entire thesis from a comprehensive point of view and provides perspectives regarding further investigations on the regulatory mechanisms and biological functions of the 2CSs herein investigated.

Table 1.1. Risk factors associated with abscess formations in K. pneumoniae infections

Risk factor Description Reference

K. pneumoniae associated factor

Phenotype attributes Hypermucoviscosity (HV) (136, 237, 272)

Extended spectrum β-lactamase (ESBL) production^a (136)

Capsular serotype (K1 or K2) (43, 67, 136, 143, 243, 267, 268)

Genetic factors

K1-specific gene magA, allS (42, 68, 136, 154)

HV regulatory gene rmpA, rmpA2 (136, 272)

Integrative and conjugative element ICEKp1 (Homologue of Yersinia high-pathogenecity island (HPI)) (145)

Iron acquisition system iucABCEiutA, iroAiroNDCB, kfu (68, 272)

pLVPK derived loci terW⁺iutA⁺rmpA⁺silS⁺ (237)

Host factor

Underlying disease Diabetes mellitus (134, 237, 239)

Urinary tract obstruction (239)

Malignancy (239)

Association with focal infections Pneumonia (239)

Urinary tract infections (239)

Therapeutic treatments Poor glycemic control (144)

Disease acquisition Community acquisition (237)

a. Negatively correlated.

10

CHAPTER 2

Molecular Characterization of the PhoPQ-PmrD-PmrAB Mediated Pathway Regulating Polymyxin B Resistance in

Klebsiella pneumoniae CG43

2.1 Abstract

The cationic peptide antibiotic polymyxin has recently been reevaluated in the treatment of severe infections caused by gram negative bacteria. In this study, the genetic determinants for capsular polysaccharide level and lipopolysaccharide modification involved in polymyxin B resistance of the opportunistic pathogen Klebsiella pneumoniae were characterized. The expressional control of the genes responsible for the resistance was assessed by a LacZ reporter system. The PmrD connector-mediated regulation for the expression of pmr genes involved in polymyxin B resistance was also demonstrated by DNA EMSA, two-hybrid analysis and in vitro phosphor-transfer assay. Deletion of the rcsB, which encoded an activator for the production of capsular polysaccharide, had a minor effect on K. pneumoniae resistance to polymyxin B. On the other hand, deletion of ugd or pmrF gene resulted in a drastic reduction of the resistance. The polymyxin B resistance was shown to be regulated by the two-component response regulators PhoP and PmrA at low magnesium and high iron, respectively. Similar to the control identified in Salmonella, expression of pmrD in K.

pneumoniae was dependent on PhoP, the activated PmrD would then bind to PmrA to prolong the phosphorylation state of the PmrA, and eventually turn on the expression of pmr for the resistance to polymyxin B. The study reports a role of the capsular polysaccharide level and the pmr genes for K. pneumoniae resistance to polymyxin B. The PmrD connector-mediated pathway in governing the regulation of pmr expression was demonstrated.

In comparison to the pmr regulation in Salmonella, PhoP in K. pneumoniae plays a major regulatory role in polymyxin B resistance.^a

a A part of this chapter has been published:

1. Cheng, H. Y., Y. F. Chen, and H. L. Peng. 2010. Molecular characterization of the PhoPQ-PmrD-PmrAB mediated pathway regulating polymyxin B resistance in Klebsiella pneumoniae CG43. J Biomed Sci 17:60-75.

2. Luo, S. C., Y. C. Lou, H. Y. Cheng, Y. R. Pan, H. L. Peng, and C. Chen. 2010. Solution structure and phospho-PmrA recognition mode of PmrD from Klebsiella pneumoniae. J Struct Biol. (In press)

2.2 Introduction

Klebsiella pneumoniae, an important nosocomial pathogen, causes a wide range of infections including pneumonia, bacteremia, urinary tract infection, and sometimes even life-threatening septic shock (196). The emergence of multiresistant K. pneumoniae has reduced the efficacy of antibiotic treatments and prompted the reevaluation of previously but not currently applied antibiotics (63, 64) or a combined therapy (116). Polymyxins, originally isolated from Bacillus polymyxa, have emerged as promising candidates for the treatment of infections (274). As a member of antimicrobial peptides (APs), the bactericidal agent exerts its effects by interacting with the lipopolysaccharide (LPS) of gram-negative bacteria. The polycationic peptide ring on polymyxin competes for and substitutes the calcium and magnesium bridges that stabilize LPS, thus disrupting the integrity of the outer membrane leading to cell death (95, 274).

The Klebsiella capsular polysaccharide (CPS), which enables the organism to escape from complement-mediated serum killing and phagocytosis (53, 113), has been shown to physically hinder the binding of C3 complement (45) or polymyxin B (30). The assembly and transport of Klebsiella CPS followed the E. coli Wzy-dependent pathway (253), in which mutations at wza encoding the translocon protein forming the complex responsible for CPS polymer translocation and export resulted in an inability to assemble a capsular layer on the cell surface (55). The CPS biosynthesis in K. pneumoniae was transcriptionally regulated by the two-component system (2CS) RcsCDB (155) where the deletion of the response regulator encoding gene rcsB in K. pneumoniae caused a loss of mucoid phenotype and reduction in CPS production (130).

In Escherichia coli and Salmonella enterica serovar Typhimurium, polymyxin B resistance is achieved mainly through the expression of LPS modification enzymes, including PmrC, an aminotransferase for the decoration of the LPS with phosphoethanolamine (125) and the pmrHFIJKLM operon (92, 260) (also called pbgP or arn operon (20, 265)) encoding enzymes. Mutations at pmrF, which encoded a transferase for the addition of 4-aminoarabinose on bactoprenol phosphate, rendered S. enterica and Yersinia pseudotuberculosis more susceptible to polymyxin B (92, 156). The S. enterica ugd gene encodes an enzyme responsible for the supply of the amino sugar precursor L-aminoarabinose for LPS modifications and hence the Ugd activity is essential for the resistance to polymyxin B (235). On the other hand, the E. coli ugd mutant with an impaired capsule also became

highly susceptible to polymyxin B (128).

The 2CS PmrA/PmrB, consisting of the response regulator PmrA and its cognate sensor kinase PmrB, has been identified as a major regulatory system in polymyxin B resistance (91, 261). The resistance in S. enterica or E. coli has been shown to be inducible by the extracellular iron (256). In addition to acidic pH (193), the role of ferric ions as a triggering signal for the expression of PmrA/PmrB has been demonstrated (261). The 2CS PhoP/PhoQ which regulates the magnesium regulon (120) could also activate polymyxin B resistance under low magnesium in S. enterica, in which the PhoP/PhoQ-dependent control is connected by the small basic protein PmrD. The expression of pmrD could be activated by PhoP while repressed by PmrA forming a feedback loop (118, 127). The activated PmrD could then bind to the phosphorylated PmrA leading to a persistent expression of the PmrA-activated genes (117).

The PmrD encoding gene was also identified in E. coli and K. pneumoniae. However, pmrD deletion in E. coli had no effect on the bacterial susceptibility to polymyxin B (256).

Recently, the PhoP-dependent expression of pmrD has also been demonstrated in K.

pneumoniae. According to the predicted semi-conserved PhoP box in the pmrD upstream region, a direct binding of PhoP to the pmrD promoter for the regulation was speculated (165).

In this study, specific deletions of genetic loci involved in CPS biosynthesis and LPS modifications were introduced into K. pneumoniae CG43, a highly virulent clinical isolate of K2 serotype (36). Involvement of the genetic determinants in polymyxin B resistance was investigated.

2.3 Results

2.3.1 Reduced production of capsular polysaccharide had minor effect on the polymyxin B resistance in K. pneumoniae

K. pneumoniae CG43 is a highly encapsulated virulent strain (36). In order to verify the role of CPS in polymyxin B resistance, the Δugd and Δwza mutants were generated by allelic exchange strategy, and their phenotype as well as the amount of CPS produced were compared with the parental strain CG43S3 and ΔrcsB mutant (130). As shown in Fig. 2.1A, the Δugd and Δwza mutants formed apparently smaller colonies on LB agar plate compared with the glistering colony of the parental strain CG43S3. Although the colony morphology of the ΔrcsB mutant was indistinguishable from CG43S3, the CPS-deficient phenotype was evident as assessed using sedimentation assay and the amount of K2 CPS produced (Fig.

2.1B). Deletion of rcsB resulted in an approximately 50% reduction of the CPS, while the Δwza mutant produced less than 20% of that of its parental strain CG43S3. The CPS biosynthesis in Δugd mutant was almost abolished, indicating an indispensible role of Ugd in CPS biosynthesis. To investigate how the CPS level was associated with polymyxin B resistance, the survival rates of the strains challenged with polymyxin B were compared. The Δugd mutant producing the lowest amount of CPS was extremely sensitive to the treatment of polymyxin B (Fig. 2.1C). Although the Δugd mutant was CPS-deficient, the impaired polymyxin resistance may have been largely attributed to the defect in LPS biosynthesis since the survival rates of Δwza and ΔrcsB mutants appeared to be comparable with the parental strain CG43S3. This argues against the notion that the level of polymyxin B resistance is positively correlated to the amount of CPS (30). Nevertheless, the possibility that a higher amount of CPS was required for the resistance could not be ruled out. As shown in Fig. 2.1D, the introduction of pRK415-RcsB (38) resulted in a significantly higher resistance to polymyxin B in both ΔrcsB mutant and its parental strain. This indicated a protective effect of large amounts of CPS in polymyxin resistance.

2.3.2 PmrF is involved in polymyxin B resistance and survival within macrophage

To investigate if the K. pneumoniae pmr homologues played a role in polymyxin B resistance, a pmrF deletion mutant strain and a plasmid pRK415-PmrF were generated. As shown in Fig. 2.2A, when the strains were grown in LB medium, a low magnesium condition (89), differences in the survival rates were not apparent. When the strains were grown in LB

supplemented with 1 mM FeCl3, an apparent deleting effect of pmrF in polymyxin B resistance was observed, and the survival rate could be restored by the introduction of pRK415-PmrF. The results indicated a role of PmrF in the polymyxin B resistance in high iron condition. In addition to the mucosa surfaces, antimicrobial peptides and proteins play important roles in the microbicidal activity of phagosome (14). To investigate the effect of pmrF deletion in the bacterial survival within phagosome, phagocytosis assay was carried out.

Since K. pneumoniae CG43S3 was highly resistant to engulfment by phagocytes in our initial experiments, the ΔrcsB mutant which produced less CPS was used as the parental strain to generate ΔpmrFΔrcsB mutant. As shown in Fig. 2.2B, deletion of pmrF resulted in an approximately four-fold reduction in the recovery rate, which was restored after the introduction of pRK415-PmrF. This indicated an important role of pmrF not only in polymyxin B resistance but also in bacterial survival within macrophage.

2.3.3 Deletion effect of Klebsiella pmrA, pmrD or phoP on polymyxin B resistance

To investigate how PmrA, PhoP and PmrD were involved in the regulation of polymyxin B resistance in K. pneumoniae, ΔpmrA, ΔphoP and ΔpmrD mutant strains were generated. Deletion of either one of these genes resulted in a dramatic reduction of resistance to polymyxin B when the strains were grown in LB medium (Fig. 2.3A). The deleting effects were no longer observed when the strains grown in LB supplemented with 10 mM magnesium, implying an involvement of the PhoP-dependent regulation in LB, a low magnesium environment. Under high-iron conditions, the deletion of pmrA caused the greatest reduction in the survival rate. Introduction of pRK415-PmrA or pRK415-PhoP into the ΔpmrAΔphoP double mutant strain not only restored but also enhanced the bacterial resistance to polymyxin B (Fig. 2.3B), which is likely due to an over-expression level of phoP or pmrA by the multicopy plasmid. Finally, whether the deletion of pmrA, phoP or pmrD affected the survival rate in phagosomes was also investigated. Interestingly, deletion of phoP resulted in most apparent effect while the pmrA deletion had less effect on the bacterial survival in macrophages. This was probably due to low iron concentration in the phagosomes (89). The introduction of pRK415-PhoP or pRK415-PmrD could restore the recovery rates of ΔphoPΔrcsB and ΔpmrDΔrcsB, although not to the extent displayed by the parental strain. Taken together, our results indicate the presence of two independent pathways in the regulation of polymyxin B resistance and the bacterial survival within macrophage phagosomes.

2.3.4 Effect of pmrA, phoP or pmrD deletion on P_pmrH::lacZ and P_pmrD::lacZ expression As the functional role of the structural gene pmrF and the regulator genes phoP, pmrD and pmrA was verified, it would be of importance to investigate the regulatory network govern by PhoPQ-PmrD-PmrAB on the expression of pmr genes. Sequence analysis has revealed PhoP and PmrA box consensus in the upstream region of pmrH and PhoP box consensus in the upstream region of pmrD (Fig. 2.4A). To investigate the interplay of PhoP, PmrA, and PmrD on the expression of pmr and pmrD genes, the reporter plasmids placZ15-PpmrH and placZ15-PpmrDwere constructed and mobilized into K. pneumoniae CG43S3ΔlacZ and its derived ΔpmrAΔlacZ, ΔpmrDΔlacZ or ΔphoPΔlacZ isogenic strains, respectively. The β-galactosidase activities of K. pneumoniae transformants under different environmental conditions were determined. In the wild-type strain CG43S3ΔlacZ, the PpmrH::lacZ activity was repressed in the presence of high magnesium but enhanced in high ferric ion (Fig. 2.4B). Such iron-inducible activity was abolished after the addition of iron scavenger deferoxamine. As shown in Fig. 2.4B, deleting effect of pmrA or phoP on the activity of PpmrH::lacZ could be observed in LB or LB supplemented with ferric iron. The negative effect of pmrD deletion was also apparent at high iron condition but was abolished after the addition of deferoxamine. The results clearly demonstrate the involvement of PmrA, PhoP and PmrD in the regulation of the expression of pmr genes, particularly in the presence of high ferric irons. As shown in Fig. 2.4C, the P_pmrD::lacZ activity was significantly reduced in high-magnesium conditions or upon the deletion of phoP. Interestingly, the deletion of pmrA or high ferric irons had little effect on the activity of P_pmrD::lacZ. The results suggest that the expression of K. pneumoniae pmrD is regulated in a PhoP-dependent but PmrA-independent manner.

2.3.5 Analysis of EMSA indicates a direct binding of the recombinant PhoP to PpmrD

The binding of PhoP or PmrA to PpmrH has been determined recently (165). In order to determine whether PhoP binds directly to PpmrD, EMSA was performed. As shown in Fig.

2.5A, binding of the recombinant His-PhoP protein to PpmrD was evident by the formation of a protein/DNA complex with a slower mobility. The binding specificity was also examined by the addition of specific competitor (PpmrD DNA) or non-specific competitor (pmrD gene or pUC19 DNA). The formation of the complex was gradually reduced with the increasing ratios of unlabeled to labeled PpmrD DNA fragments and completely diminished at the highest ratio investigated. In the presence of unspecific competitor DNA, the formation of

protein/DNA complexes could also be observed although a portion of free probe was still noted, possibly due to the high amounts of DNA added. As shown in Fig. 2.5B, the formation of protein/DNA complex diminished when His-PhoPN149, in which the carboxyl-terminal helix-turn-helix domain has been truncated, was used instead of His-PhoP. The results strongly suggest the PhoP binds via its C-terminal domain to the promoter of pmrD for the activation of the pmrD expression in K. pneumoniae.

2.3.6 Two-hybrid analysis of the in vivo interaction between Klebsiella PmrD and PmrA The interaction between Klebsiella PmrD and PmrA has been shown as a prerequisite for the connector-mediated pathway (165). To demonstrate in vivo interaction, a bacterial two-hybrid assay was performed. The plasmid pBT-PmrA carrying the RNAPα-PmrA coding region and the plasmid pTRG-PmrD carrying the λ-cI-PmrD coding sequence were generated.

In vivo interaction between the two reporter strains allowed the binding of λ-cI to the operator region as well as the recruitment of α-RNAP for the expression of the ampR and lacZ reporter genes. The bacteria harboring the positive control plasmids pTRG-Gal11^P and pBT-LGF2 showed a vigorous growth on the indicator plate, as reflected by the apparent colony formation when the culture was diluted serially (Fig. 2.6A). In contrast, the strain carrying the negative control vectors pBT and pTRG revealed impaired colony formation. As shown in Fig. 2.6A, a profound growth pattern of the E. coli cells harboring pBT-PmrA and pTRG-PmrD was observed indicating an interaction between the PmrD and PmrA.

2.3.7 PmrD prevents the dephosphorylation of PmrA catalyzed by PmrB

In S. enterica, the phosphorylation of PmrA by the cognate sensor protein PmrB has been demonstrated to enhance its affinity in binding to its target promoter. The subsequent dephosphorylation of PmrA by PmrB helped to relieve from over-activation of this system (1). In Salmonella, PmrD has been shown to be able to protect PmrA from both intrinsic and PmrB-mediated dephosphorylation (22). To verify if Klebsiella PmrD also participates in the phosphorylation, in vitro phosphotransfer assay was carried out with the recombinant proteins

In S. enterica, the phosphorylation of PmrA by the cognate sensor protein PmrB has been demonstrated to enhance its affinity in binding to its target promoter. The subsequent dephosphorylation of PmrA by PmrB helped to relieve from over-activation of this system (1). In Salmonella, PmrD has been shown to be able to protect PmrA from both intrinsic and PmrB-mediated dephosphorylation (22). To verify if Klebsiella PmrD also participates in the phosphorylation, in vitro phosphotransfer assay was carried out with the recombinant proteins