Proteus Toxic Agglutinin, a Novel

Autotransported Cytotoxin with no

Bacterial Homologs

Praveen Alamuri
Mobley Lab
812 Seminar
 Outline
• Proteus mirabilis and complicated urinary tract infections

• Autotransporter proteins

• Proteus Toxic Agglutinin (Pta)

• Characterization of autotransporter Pta

• Role of Pta in P. mirabilis

• Pta as a vaccine candidate

• Summary and Future directions

 Proteus mirabilis: The “Sticky” Bug

• Enterobacteriaceae: Swimmer Cell Swarmer Cell

Gram negative rod, Motile

• 2 x 0.5 µM in size

• Dimorphic

• Normal flora of gut

• Opportunistic uropathogen

• Efficient biofilms

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kPUB/chaphtm/333/01_00.htm
Proteus Complicated Urinary Tract Infections

• Long-term catheterization

• Wound borne infections

• Resistant to antibiotic therapy Proteus swarming rafts on

silicone catheters

• Polymicrobial infections

• Rising treatment costs

• Efforts to develop vaccines Catheter obstruction

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 Pathogenesis of Proteus Infections
Ascending urinary tract infection Established virulence factors
bacteremia

pyelonephritis
?
Hemolysin IgA-protease

?
Urease
Fe2+

Flagella
cystitis Fimbriae
Irreversible tissue damage

Unidentified virulence factors predicted

 Genome Sequence Provides New Hints

• Catheter-associated strain HI4320

• 4.3 Mb comprising 3685 ORFs
• Type III system: no role in UTI

Pearson et al., (Unpublished)

Six putative autotransporters (AT)

• PMI 0844, 2176, 2341 (pta): Protease-like
• PMI 2122, 2174, and 2575: Adhesin or hemagglutinin-like
 Autotransporters: Simple yet Elegant
Autotransporter pathway
N

• In pathogenic Gram-negatives
β OM • Surface expressed proteins
C
N
C
• Long signal sequence
PP
• N-terminal alpha: trait / function
Sec-system
• C-terminal beta: translocation
IM

Primarily virulence factors

SS Alpha/Passenger Beta
N C
 Functional Diversity of Autotransporters
Cell surface-associated alpha Released alpha
OM Outside OM Outside

Adhesion

Autogglutination and
biofilm

Serum resistance Cytotoxicity

Hemagglutination

Effective Antigens in Vaccines

Pta: Immunogenic Putative Serine Protease AT
2339 2340 pta 2342
Transposase Sensor His Kinase Serine protease AT Putative Copper
3.2 kb oxidase
α β

1 58 H147 S366 D533 730 1072

117 kDa

N 9 anti-parallel C
β sheets

• The 3.2 kb gene encodes a 117 kDa protein

• Alpha domain resembles Subtilisin-clan of serine proteases

• Beta domain resembles translocator domain and ends with “YFW”

Rationale Behind the Characterization of Pta

• Subtilisins are important proteases in cellular functions

• Very few non-Bacillus subtilisins studied in bacteria

• No functional or structural homologs

• Immunogenic outer membrane protein in P. mirabilis

Pta

P. mirabilis

Nielubowicz and Mobley

 Pta has No Bacterial Homologs
• Only ~15 % identity with other serine proteases
• Ser366, His147, and Asp533 make the catalytic triad
• Identity restricted to regions spanning catalytic domains

Catalytic His Catalytic Ser Catalytic Asp

147 156 364 374 531 540
*
PmirabilisPta HGTHVTGTVG GTSMAAPHVTG AKDAGLYDGH
STECSubA HGTAMASLIA GTSEATAIVSG LTDKNTPVVN
FpennFls HGTHVAGTIA GTSMAAPHVTG IIDDFGGLNN
PschloroPspA HGTHVTGTMG GTSMAAPHATG ILDDGGILVD
Consensus HGSXVSGXLS GTSXSXPXXSA LLDDGLXXXD
subtilase T ITS IT A TG II S I N
M C A VA A VV T V H
G MG V MM A M
C AC C F F
L A C
V
 Cn3D Based Fold Prediction of Pta

Fervidolysin From Fervidobacterium pennivorans,

A Keratinolytic Enzyme Related To Subtilisin : ~ 20 % identity

Red = Identical amino acids

Yellow = Active Site
Ser 366
Asp 533

His 147

Ca++ binding site

 Hypothesis

The putative autotransporter Pta either directly or indirectly

contributes to virulence in P. mirabilis

Goals:
• Determine the function of Pta in E. coli
• Characterize passenger domain
• Assign a role for Pta in P. mirabilis
• Test its efficacy as a vaccine
 Various Forms of Pta Used in this Study
α β

1 58 H147 S366 D533 730 1072

Pta
N C

1 58 H147 S366A D 533 730 1072

Pta*
N C

1 58 H147 S366 D 533 730

Pta-α
N C

1 58 H147 S366A D 533 730

Pta-α *
N C
Purification and Initial Characterization
in E. coli
 Pta is 110 kDa Cell Surface-Associated AT
T7 OM
Adhesin ?
pta Agglutinin ?
C-term.6X His-tag in
E. coli BL21plysS WHOLE CELL PROTEIN OUTER MEMBRANE PURE
CTL Pta Pta*
Inner membrane: Pta Pta* CTL Pta M KDa
I U I U I
Sarkosyl 250
150
100
Outer membrane: 75
Zwittergen 50
37

Ni++-NTA–Imidazole
25
20
On-column
refolding and elution
 Interaction of E. coli-Pta with Hep2

37 ºC; 45 min Giemsa stain

Image

E. coli Hep2

E. coli - Vector E. coli - Pta E. coli - Pta * E. coli - Ptaα

Evidence of Evidence of
cytotoxicity autoaggregation 100X; Bar=100 µm
 Quantitative estimation of cell lysis
Cytotox-one Membrane integrity assay

Leaky cell
LDH
LDH
Lactate Pyruvate
NAD+ NADH

Resorufin Resazurin

Pure Pta or Estimate LDH in

Pta * culture supernatant
Bladder Cells
(PBS and 5% Triton)
 Kinetics of Bladder Cell Lysis by Pta

100 8 µg/ml
% Maximum Lysis

6 µg/ml
80
4 µg/ml
60
2 µg/ml
40
Pta *
20
8 µg/ml
0
0 30 60 90 120
Minutes

Maximum lysis with 8 µg/ml for 120 mins

 Qualitative Estimation of Bladder Cell Lysis
PBS 0 min 30 60 90 120

Pta 8 µg/ml

Pta *:
8 µg/ml - 120 mins

60X ; Bar=100 µM
• Membrane-associated 110 kDa AT
• Indication of cytotoxicity and agglutination

Characterization of the Purified Pta-α

Purification of the Passenger Domain: Pta-α
1 58 147 366 533 735

N His or Ala Ser or Ala Asp C

Cell Protein SOL. OM

CTL α CTL α Pta- α M CTL α

~75 kDa Pta-α purified from the soluble fraction

 Protease Activity of Pta-α
**
12 **

hydrolyzed/min/mg protein
PTA 
[s] = 100 uM 10
PTA  (S366A)

 mol substrate
[E] = 20 nM 8 PTA  (H147A)
PTA  -PMSF
HEPES 6 Buffer
buffer pH 8.0 4
20 mM Ca2+ **
2

0
S1 S2 S3
Substrate
S1 = N-Suc-Ala-Ala-Pro-Phe-
pNa Chymotrypsin-like
S2 = N-Suc-Ala-Ala-Pro-Leu-pNa
S3 = N-Suc-Ala-Ala-Pro-Lys-pNa Trypsin-like
 Pta is an Alkaline Protease

S1 = 100 µM
Pta-α = 20 nM
Pta-α pre-incubated
at various pH

S1 = 25 - 150 µM
Pta-α = 20 nM 0.087
1/ v
pH = 8.5

KM = 22 µM - 0.045

1/[s]
 Pta is a Subtilisin-like Alkaline Protease
20 nM Pta-α 100 µM S1 Protease activity
estimated
Incubated with pH 8.5
inhibitor
Inhibitor % Inhibition

PMSF 90
Serine protease
DFP 95
TLCK 0
TPCK 25 Non-trypsin like
STI 80
BBI 75 Subtilisin-like
Chymostatin 90
Subtilisin-like
Leupeptin 100
 Cytotoxicity Attributed to Protease Activity
37 ºC; 2 hrs Quantify
LDH
release in
Purified Pta-α Cultured host culture
8 µg/ml cells supernatant
120 ** PTA
** PTA *
% Maximum Lysis

100 PMSF-Treated
(PTA ) **, P<0.01
80

60

40

20

0
HEK293 UMUC-3 VERO BHK21 MMK.5
Cell Line
 Actin and Nuclear Damage by Pta
Phalloidin DAPI Merge

PBS
Vero Cell
Monolayer
Pta-α
Pta-α 8 µg/ml
for 45 mins

Stain with FITC- Pta-α*

Phalloidin
Pta-α
DAPI Stain
PMSF-
treated
100X; Bar=100 µM
 Four Hours Far Too Much

ACTIN DAPI MERGE

PBS
Treated

Pta
8 µg/mL
for 4 hrs

100X; Bar=100 µM
• Pta is a 110 kDa subtilisin-like alkaline protease AT
• Cell surface-associated cytotoxin
• Function associated with the passenger domain
• May be an agglutinin

Role of Pta in P. mirabilis

 Pta is an Inducible Autotransporter
25º 37º 42 º St Exp

P. mirabilis cultured
under different
conditions
pH
kDa M 5 9 7
Outer membrane 150
fraction collected 100

Immunostained with Mg2+ Ca2+

Anti-Pta antisera mM 0.5 1 2 0 20
 Inactivation and Complementation of pta
aphA3
5’ Alpha domain Beta domain 3’

ParaC

WT pta
pta

OD600
pBAD
a
-pt
A D AD
T / pB / pB
a a a
W pt pt pt
250
Hours
150

100 Independent cultures

75 in LB at 37 °C
 Pta Contributes to Cytotoxicity in P. mirabilis
Washed
bacteria
37 ºC; 3 hrs Quantify
LDH
release
P. mirabilis Bladder cells
5
LB + Glycerol + 10 bacteria
Ca 2+ pH 8.5
** 6
100
% Maximum Lysis

10 bacteria
7
10 bacteria
80 **
60

40 * **
20

* P<0.05 0
** P<0.01 WT WT/ pta - pta- / pta-/
pBAD-pta pBAD-pta pBAD-pta*
 Pta Promotes Autoaggregation of P. mirabilis
Cells in buffer
pH 8.5 600

t0 = ~460 Kletts 500

Mean Klett Units

400 *
pta -
300
WT
200 pta/pBAD-pta*
100 pta/pBAD-pta
t = 3 hr
WT/pBAD-pta
Drop in Cell density 0
in the suspension 0 60 120 180 240 300
Minutes

Autoaggregation is independent
of protease activity
Autoaggregation
 Mutant Attenuated in Kidney and Spleen
Independent Challenge WT HI4320 pta - N=10

WT pta
P=0.10 P=0.06 P=0.007 P=0.03
10
5 x 10 CFU Log10 CFU/g tissue or ml
7

urine 8

4
Plate
2
homogenates
after 7 days 0
Urine Bladder Kidney Spleen
 Role for Pta in Virulence

Alkaline urinary tract

Surface
expression of Pta
Autoaggregation

Host cell
membrane
disruption

Host cell
lysis
Pta as a Vaccine Against P. mirabilis

An ideal vaccine candidate:

• Surface exposed

• Stable expression in the host

• Important for virulence

 Immunization with Pta*

Naïve : Cholera toxin

4-6 week
old CBA/J Immunized: Pta* + Cholera toxin

Intranasal Immunization

Days 0 7 14 21 28
Immunize Boost Challenge Sacrifice
50 µg Pta*+ 5 µg CT 25 µg Pta*+ 5 µg CT 5x107 CFU
5 µg CT 5 µg CT P. mirabilis

Collect Pre-immune sera Collect Post-immune sera

 0.0448 0.001 0.0017

0.036 0.0002 0.0006 CT

Pta
Pta-α

N = 10

1° = 50 µg
Boost = 20 µg

BLADDER KIDNEY SPLEEN

• Immunization with either Pta or Pta-α offers moderate to significant

protection against Proteus infections
• Surprisingly better protection with Pta than with Pta- α
• The difference in between them is not significant
Immunization with Pta* : Complete data
0.041 <0.001 0.0015 1° = 50 µg
Boost = 20 µg
N = 24

Future Directions
• ELISA IgG, IgM, IgA
• Try IV or IP Route
• Continue with Pta-α

CT Pta CT Pta CT Pta

Bladder Kidney Spleen

 Pta-Specific Ig Titer Correlates with Protection

P<0.0001
Measure at A405 N=6
P<0.01
Goat
Anti-Mouse
Ig-AP

Pre- or Post
Immune Sera

P=0.03
Pta Binding

Indirect ELISA Pre Post Pre Post

Cholera Toxin Pta*

1:512 titer evaluated
 Pta, a New Virulence Factor in P. mirabilis

Hemolysin IgA-protease

Urease Fe 2+
Acquisition

Flagella
Fimbriae
Pta

• Autotransporters in P. mirabilis : New avenue of research

• Surface-associated cytotoxin : New concept in pathogenesis
• Significance of physiological conditions in studying virulence
 Functional Diversity of Autotransporters
Cell surface-associated alpha Released alpha
OM OM Outside
Outside

Adhesion

Autoagglutination
Cytotoxic
Serum resistance

Hemagglutination

Effective Antigens in Vaccines

 Future Directions

• Determine targets for Pta in host cells

Pull down assays, host cell membrane staining
• Histology studies using mouse model
WT, pta-, hpmA-, pta-/hpmA-
• Explore the autoaggregation phenotype
• Immunization using other adjuvant and additional controls
• Characterize other autotransporters in P. mirabilis
Potential non-fimbrial adhesins
 Thanks are due to….
Dr. Harry Mobley
Mobley Mob

Stephanie Himpsl
Sara Smith

Sam Straight Department

NIH
Been there…done that…

Freaked out

www.promega.com
 HeLa Infected with Pta
Buffer + 0.20 % ZW Buffer + 0.35 % ZW

5 ug of 2341 for 2 hrs 10 ug of 2341 for 2 hrs Heat inactivated

10 ug
 Mutant Sensitive to Pta

10
HI3420
Log10 CFU/ml
8
6 pta
4
Co culture
2
0
1 2 3 4 5 6 7 8 9 10 11
Hours
 Wild-type Out-competes pta Strain in vivo
P=0.13 P=0.02 P=0.0023 P=0.0018
WT : pta
1 1

5 x 107 CFU

Plate
homogenates
Urine Bladder Kidney Spleen
after 7 days
 Goals of the Study
Cytotoxic protease

Surface adhesin

Ability to confer immune protection

 Characterization of the Beta-Domain

OM Hydrophobic beta sheet

Hydrophilic loop

1 α 844 910 1016 1054 1072

Sites of C-terminus truncations

T-844 T-910 T-1016 T-1054 Pta

M CE OM CE OM CE OM CE OM OM
250
150
100
 Beta domain essential for protein localization at the OM
1 α 730 844 910 1016 1054

Sites of C-terminus truncations

CELL EXTRACT SOLUBLE OUTER MEMB

CTRL α CTRL α PURE α M CTRL α

• Purified α-domain from the
* soluble fraction of the cell
• Alpha domain not seen in OM

T- 844 T- 916 T-1016 T-1044 PSPC

M CE OM CE OM CE OM CE OM OM
250
150
100

75
 Swim Swarm

WT

pta
 Urolithiasis: Hallmark of Proteus Infections

pH
Urea NH4+ +
HCO3

NH3 +
Urease CO2
Stones in an infected
mouse bladder

Precipitation of Ca2+ and Mg2+

Bacteria inaccessible to antibiotics
Catheter obstruction
 Approach to Identify Candidate Antigens

An ideal vaccine candidate:

• Surface expressed
• Stable expression in the host
• Important for virulence or persistence

Pta

P. mirabilis
Nielubowicz and Mobley
Immunization with Pta
Offers Moderate Protection

P=0.0891 P=0.0001 P=0.0467 CT

CT + Pta*

N=12

Bladder Kidney Spleen

 Proteus Infection: Irreversible Tissue Damage
Urease Hemolysin (HpmA)
NH3 + CO2
Whole cells or
Urea culture supernatants are
cytotoxic and hemolytic
Toxic to epithelial cells

Catheterization aggravates
urolithiasis

hpmA- mutant still cytotoxic in vivo

Unidentified virulence factors predicted