Proteus mirabilis

The investigation of RpoN in swarming of uropathogenic Proteus mirabilis

Shwu Jen Liaw, Ph.D.

2017/10/23 Department and Graduate Institute of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan

1

Introduction

2

Proteus mirabilis • Gram-negative, facultative anaerobic Enterobacteriaceae • Urinary tract infection (UTI) - catheter-associated urinary tract infections - urolithiasis - pyelonephritis

By Bobby Henderson

Wound infection, septicaemia, pneumonia

Coker et al. Microbes Infect. 2000. 2: 1497–1505.

3

Proteus mirabilis • Virulence factors

Fimbriae: Mrp and P-fimbriae Haemolysin Urease IgA protease Flagella • Swarming short rod swimmer cell/ elongated multicellular swarmer cell 4

Four separate phases in swarming of P. mirabilis    

Induction of swarmer cell differentiation Delay prior to onset of swarming Active swarming migration Consolidation

Swarming motility

Soft Matter, 2009, 5, 1174-1187

Coordination of virulence factor expression and swarming migration of P. mirabilis

6

P. mirabilis regulatory networks of swarming and virulence Flagella Lipopolysaccharid e

Chemotaxis Two-component

Trends in Microbiol 16:496 2008

7

Results

Tn5 - mutagenesis S17-1

E. coli S17-1

P. mirabilis N2

Kmr Tets

Tetr Kms

10 mM MgSO4

conjugation

37℃/overnight

(on LSW- agar)

10 mM MgSO4

Km 50μg/ml

Plating on selection agar

Amp 50μg/ml

37℃/overnight

Km 50 μg/ml Tet 12.5μg/ml

9

RpoN regulates swarming

RpoN • A bacterial RNA polymerase alternative sigma factor • RpoN has been shown to play a key role in stress responses (N limitation). • -24, -12 element NOT -35, -10 element (RpoD)

Nucleic Acids Res. 2014 Dec 1;42(21):12961-72.

• A master regulator of flagellin synthesis

XylE activity of flhDC promoter

No effect of RpoN on the promoter activity of flhDC

3

WT

ΔrpoN

5

7

Time (h)

24

RpoN affects the mRNA levels of flhDC and fliC

Why can RpoN affect mRNA levels of flhDC and fliC ?

SrY regulates swarming motility

Bacterial small RNAs (sRNAs) • small non-coding RNA molecules • sRNAs usually regulate gene expression posttranscriptionally by binding to mRNA targets.

Viegas, Arraiano. RNA Biol 5 (4): 230–43 (2008).

16

SrY regulates expression of cheA

CheA: a chemotaxis kinase involved in flagellar assembly. The chemotaxis system plays an essential role in swarm cell differentiation and motility

SrY regulates expression of flhDC

SrY regulates expression of rcsB RcsB: a bacterial two-component response regulator negatively regulating P. mirabilis swarming by downregulation of flhDC

Liaw et.al.

SrY small RNA

May RpoN affect swarming through SrY ?

RpoN regulates expression of srY

Conclusions

Conclusions • RpoN positively regulates SrY. • SrY negatively regulates rcsB mRNA level and hence flhDC. • SrY positively regulates cheA

Thanks for your attention ~

25

Salmonella flagella

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Bacterial two component system

Putative RpoN-swarming regulatory network in Proteus mirabilis N2

?

?

RpoN modulates glnA, glmY or flhDC expression 160

xylE

pACYC184 pyrocatechol

OD375

N2

ΔrpoN

30 25 20 15 10 5 0

Wt ΔrpoN

3 5 7 24 flagellin

Time (h)

N2

ΔrpoN

120

6

Relative fold change

promoter

35

XylE activity of glmY-qseE promoter

Transcriptional fusion

XylE activity of glnA promoter

40

qPCR

7

80

40

0 3 5 7 24 Time (h)

wt

△rpoN

com

5 4 3 2 1 0 glnA glmY flhDC

sRNA GlmY regulates cheA mRNA 1.4 1.2

1.4 wt 1.2

glmY

1

Translational fusion reporter

XylE activity

XylE activity

1 0.8 0.6

0.8 0.6

0.4

0.4

0.2

0.2

0

wt glmY

cheA

0

cheA