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Identification of Outer Membrane Proteins

Identification of Outer Membrane Proteins

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01/30/2013

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Identification of outer membrane proteins of 
 Mycobacterium tuberculosis
Houhui Song
a,d
, Reatha Sandie
b,d
, Ying Wang
a,d
,Miguel A. Andrade-Navarro
b,c
, Michael Niederweis
a,
*
a
Department of Microbiology, University of Alabama at Birmingham, 609 Bevill Biomedical Research Building,845 19th Street South, Birmingham, AL 35294, USA
b
Bioinformatics Group, Ottawa Health Research Institute, 501 Smyth Road, Ottawa, Ontario, K1H 8L6, Canada
c
Max Delbru¨ ck Center for Molecular Medicine, Robert-Ro¨ ssle-Str. 10, 13125 Berlin, Germany 
Received 13 September 2007; received in revised form 8 February 2008; accepted 18 February 2008
KEYWORDS
Secondary structure;Prediction;Amphiphilicity;Beta-strand;Exported;Inner membrane;Periplasmic;Secreted proteins
Summary
The cellwall of mycobacteria includes an unusualouter membraneof extremely low permeabil-ity. While
Escherichia coli
uses more than 60 proteins to functionalize its outer membrane, onlytwo mycobacterial outer membrane proteins (OMPs) are known. The porin MspA of
Mycobacte-rium smegmatis
provided the proof of principle that integral mycobacterial OMPs share the
b
-barrelstructure,theabsenceofhydrophobic
a
-helicesandthepresenceofasignalpeptidewithOMPs of gram-negative bacteria. These properties were exploited in a multi-step bioinformaticapproach to predict OMPs of
M. tuberculosis
. A secondary structure analysis was performed for 587 proteins of
M. tuberculosis
predicted to be exported. Scores were calculated for the
b
-strand content and the amphiphilicity of the
b
-strands. Reference OMPs of gram-negative bac-teria defined threshold values for these parameters that were met by 144 proteins of unknownfunctionof
M.tuberculosis
.TwoofthemwereverifiedasOMPsbyanoveltwo-stepexperimentalapproach. Rv1698 and Rv1973 were detected only in the total membrane fraction of
M. bovis
BCGinWesternblotexperiments,whileproteinaseKdigestionofwholecellsshowedthesurfaceaccessibility of theseproteins. Thesefindings established that Rv1698 and Rv1973 are indeedlo-calized in the outer membrane and tripled the number of known OMPs of
M. tuberculosis
. Sig-nificantly, these results provide evidence for the usefulness of the bioinformatic approach topredict mycobacterial OMPs and indicate that
M. tuberculosis
likely has many OMPs with
b
-bar-rel structure. Our findings pave the way to identify the set of proteins which functionalize theouter membrane of
M. tuberculosis
.
ª
2008 Elsevier Ltd. All rights reserved.
 Abbreviations:
OM, outer membrane; OMP, outer membrane protein; IM, inner membrane; IMP, inner membrane protein; wt, wild-type.* Corresponding author. Tel.:
þ
1 205 996 2711; fax:
þ
1 205 934 9256.
E-mail address:
mnieder@uab.edu(M. Niederweis).
d
These authors contributed equally to this work.1472-9792/$ - see front matter 
ª
2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.tube.2008.02.004
available at www.sciencedirect.comjournal homepage:http://intl.elsevierhealth.com/journals/tube
Tuberculosis (2008)
88
, 526
e
544
 
Introduction
The cell wall of mycobacteria is an intriguingly complexstructureconsisting of a great variety and a large amountof lipids.
Very long-chain fatty acids, the mycolic acids,are covalently bound to the arabinogalactan-peptidoglycanco-polymer and were proposed to form the inner layer ofan asymmetric outer membrane while other lipids consti-tute the outer leaflet.
3
X-ray diffraction studies showedthat the mycolic acids are indeed oriented in paralleland perpendicular to the plane of the cell envelope.
4
The presence of a second lipid bilayer outside of thecytoplasmic membrane in mycobacterial species hasrecently been visualized for the first time in a near nativestate by cryo-electron microscopy.
5
The discovery and theanalysis of the porin MspA of
Mycobacterium smegmatis
provided the first conclusive evidence that functionallysimilar, but structurally completely different outemembrane proteins (OMPs) exist also in mycobacteria.
6
e
9
Despite the well-documented importance of OMPs for theimport of nutrients, secretion processes andhost
e
pathogen interactions in gram-negative bacteria,
surpris-ingly few OMPs of mycobacteria are known. The only twowell-characterized examples of integral OMPs are theporin MspA of
M. smegmatis
andthechannel-formingprotein OmpA of
M. tuberculosis
.
e
By contrast,
E.coli
uses mor e than 60 proteins to functionalize its outer membrane,
none of which has significant sequencesimilarity to any
M. tuberculosis
protein.Traditionally, OMPs have been discovered by isolatingthe cell envelope and then separating the inner from theouter membrane in sucrose gradients.
e
Due to thecovalent linkages between the peptidoglycan, the arabi-nogalactan and the mycolic acid layer,
1
it is difficult tomechanically lyse mycobacterial cells.
This is usuallyachieved only by harsh conditions, which inadvertentlyleads to mixing of components of both membranes. Thishas hampered localization experiments and identificationof
M. tuberculosis
OMPs so far.
Bioinformatic analysis ofthe genome of
M. tuberculosis
provides an alternativestrategy, but OMPs are more difficult to identify by theamino acid sequence than inner membrane proteins,whose hydrophobic
a
-helices are predicted with accura-cies exceeding 99%.
So far, all known OMPs are
b
-barrelproteins, which are characterized by a pattern of alter-nating hydrophobic and hydrophilic amino acids in the
b
-strands forming the
b
-barrel.
Such a pattern is recog-nizable in the protein sequences and has been exploitedin recent years to develop programs for prediction of
b
-barrel proteins. For example, more than 10 previouslyunknown OMPs were predicted for 
E. coli
.
Notably,a consensus method performed better than each individ-ual prediction method for a set of 20
b
-barr elOMPs whosestructures are known at atomic resolution.
The successof these approaches motivated the application of oneofthese algorithms to predict OMPs of
M. tuberculosis
.
However, the usefulness of this analysis is limited for several reasons: (i) Pajon et al. did not exclude proteinswith hydrophobic
a
-helices and therefore the list of pre-dicted OMPs contains a large number of inner membraneproteins (IMPs). (ii) One of the two variables chosen aspredictors of putative
b
-barrel structures wasbased onthe prevalence of a C-terminal phenylalanine,
which istypical for OMPs of gram-negative bacteria but is notpresent in the two known mycobacterial OMPs MspA andOmpA.
(iii) Proteins with sequence homology toknown cytoplasmic and periplasmic proteins or lipopro-teins, which are anchored in membranes by their lipidmoieties and not by a transmembrane
b
-barrel,
werenot excluded from the list.In this study, we have employed an algorithm entirelybased on physical principles to predict OMPs of
M. tubercu-losis
. In contrast to all other prediction methods so far, weprovide a scoring system for the number and amphiphilicityof
b
-strands of a particular protein. By combining bothparameters with biological knowledge we predicted 144proteins as OMPs of
Mtb
. The subcellular localization oftwo of these proteins was determined by demonstratingtheir association with membranes and their surface acces-sibility to proteases. This alternative approach identifiedRv1698 and Rv1973 as OMPs of
M. tuberculosis
and providedexperimental evidence for the usefulness of the bioinfor-matic predictions.
Materials and methods
Genome-wide analysis to identify putativeOMPs of 
M. tuberculosis
A FASTA file with 3991 protein sequences for the
Mycobacte-rium tuberculosis
H37Rv genome was obtained fromftp://ftp.ncbi.nih.gov/genbank/genomes/Bacteria/Mycobacter ium_tuberculosis_H37Rv/[version: AL123456.2 date: 04/18/2006].
All sequences were scanned for sequence simi-larity matches against the Pfam database of protein motifs(version 20.0
) using hmmpfam.
To identify exportedproteins of
M. tuberculosis
we predicted thepresence ofan N-terminal signal peptide using SignalP 3.0.
All pro-teins that had a
max
score
!
0.51 were considered asexported proteins. These proteins were selected and thesequences corresponding to the signal peptide predictedby SignalP were removed. These shortened sequencesshould represent the mature proteins and were used for allfurther analyses. Next, the sequences were examined byTMHMM to predict transmembrane
a
-helices.
For allproteins that did not have a hydrophobic
a
-helix the second-ary structurewas predicted from the sequence using theJnet algorithm
which gives the best performance amongsecondary structure prediction algorithms and achievesa 76.4% average accuracy on a large test set of proteins.
Predicted
b
-strands of a minimum of five consecutive resi-dueswereregistered.Next,wecomputedtheamphiphilicityof these
b
-strands. To this end, the mean hydrophobicity ofone side of a
b
-strand
b
(
i
) was calculated at position
i
ina sequence following Vogel and Ja¨hnig
as
b
(
i
)
Z
1/5
Â
(
h
(
i
À
4)
þ
h
(
i
À
2)
þ
h
(
i
)
þ
h
(
i
þ
2)
þ
h
(
i
þ
4)), where
h
(
i
)is the hydrophobicity of the amino acid at position
i
. Notethat in a sequence of amino acids from 1 to
, these valuescan only be computed for 
i
Z
5,
.
,
À
4. The values ofhydrophobicity for the amino acids were taken from Sweetand Eisenberg.
Given the average value of hydrophobicityOuter membrane proteins of
M. tuberculosis
527
 
overawholesequence
b
m
,wecountedthezerocrossingsofthe
b
e
b
m
. We combined this measurement with the sec-ondary structure prediction. The number of hydrophobicitycrossings per residues in
b
-strand was defined as ‘‘amphiphi-licity’’ of the
b
-strands of a protein and used as discriminat-ingparameter.Highervaluesindicatethepropensitytoformatransmembrane
b
-barrel(Figure1).Further,thenumberofcysteines was counted, and the isoelectric point of the pro-tein was computed using the piCalculator BioPerl module(http://www.bioperl.org/).
Analysis of the secondary structure of selected proteins
The SignalP3.0 algorithm
was accessed athttp://www.cbs.dtu.dk/services/SignalP/toconfirmthepresenceofsig-nal peptides for selected proteins of
M. tuberculosis
. TheTMHMM program
and the ConPred II prediction server 
were used for prediction of hydrophobic transmembrane
a
-helices.
M. tuberculosis
proteins were considered not tobe integral inner membrane proteins when both methodsdid not predict a transmembrane
a
-helix. All programswere used with standard settings unless otherwise noted.
Bacterial strains and growth conditions
Mycobacterium smegmatis
mc
2
155 was grown at 37
C inMiddlebrook 7H9 liquid medium (Difco Laboratories) sup-plemented with 0.2% glycerol, 0.05% Tween 80 or onMiddlebrook 7H10 agar (Difco Laboratories) supplementedwith 0.2% glycerol unless indicated otherwise. For growthof
Mycobacterium bovis
BCG ATCC 27291 the enrichmentOADC (BD Biosciences) was added to the Middlebrook 7H9liquid medium.
Escherichia coli
DH5
a
and
Escherichia coli
Rosetta (Novagen) were used for cloning experiments andfor overexpression of
ompA
,
rv1698 
and
rv1973
, respec-tively, and were routinely grown in LB medium at 37
C.The following antibiotics were used when required at thefollowing concentrations: ampicillin (100
m
g ml
À
1
for 
E.coli
), kanamycin (30
m
g ml
À
1
for 
E. coli
; 10
m
g ml
À
1
for mycobacteria), hygromycin (200
m
g ml
À
1
for 
E. coli
, 50
m
gml
À
1
for mycobacteria).
0.00.20.40.60.81.00.0 0.1 0.2 0.3 0.4 0.5 0.6
    a    m    p     h     i    p     h     i     l     i    c     i     t    y
0.20.30.40.50.60.70.80.91.00.1 0.2 0.3 0.4 0.5
    a    m    p     h     i    p     h     i     l     i    c     i     t    y
Rv1698Rv1973MspAOmpA/Rv0899
AB C
587exported proteins3991
Mtb
proteinsSignalPTMHMMno homologs of other subcellular locations144putative OMPs
β
-strand contentamphiphilicity299 proteins withamphiphilic
β
-strands453 proteinswithoutTM
α
-helix
134 IMPsunknown OMPsPE-PGRSMcePPEexported
Mtb
proteinsreference proteins
β-strand content β-strand content
Figure 1
Prediction of putative OMPs of
M.tuberculosis
H37Rv. (A) Strategy.The 3991 predicted proteins of
M.tuberculosis
H37Rvwere analyzed for the presence of asignal peptide using the SignalP algorithm.
The 587 proteins containing a signal peptidewereanalyzed using the TMHMM algorithm
to recognize hydrophobic
a
-helices. Their 
b
-strand content was predicted using Jnet.
Theamphiphilicity of the
b
-strands was computed using an algorithm from Vogel and Ja¨hnig.
(B)
b
-strand content and amphiphilicity ofthe predicted exported proteins. The minimal length of a transmembrane
b
-strand was set to five amino acids. The red diamondsrepresent 29 known OMPs from gram-negative bacteria and from mycobacteria (MspA, OmpA/Rv0899). (C)
b
-strand content andamphiphilicity of 144 putative outer membrane proteins. Only exported proteins are depicted that do not have a transmembrane
a
-helix and have
b
-strand content of at least 0.09 and amphiphilicity of at least 0.19. In addition, all proteins with similarities to pro-teinswithothersubcellularlocalizationsarenotshown.Rv1698 andRv1973werechosentoexperimentallyexaminetheirsubcellular localization. The purple, orange and green diamonds represent members of the PE-PGRS, Mce and PPE families, respectively.
528 H. Song et al.

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