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From Evolution to Pathogenesis: The Link Between β-Barrel Assembly Machineries in the Outer Membrane of Mitochondria and Gram-Negative Bacteria

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β-barrel proteins are the highly abundant in the outer membranes of Gram-negative bacteria and the mitochondria in eukaryotes. The assembly of β-barrels is mediated by two evolutionary conserved machineries; the β-barrel Assembly Machinery (BAM) in Gram-negative bacteria; and the Sorting and Assembly Machinery (SAM) in mitochondria. Although the BAM and SAM have functionally conserved roles in the membrane integration and folding of β-barrel proteins, apart from the central BamA and Sam50 proteins, the remaining components of each of the complexes have diverged remarkably. For example all of the accessory components of the BAM complex characterized to date are located in the bacterial periplasm, on the same side as the N-terminal domain of BamA. This is the same side of the membrane as the substrates that are delivered to the BAM. On the other hand, all of the accessory components of the SAM complex are located on the cytosolic side of the membrane, the opposite side of the membrane to the N-terminus of Sam50 and the substrate receiving side of the membrane. Despite the accessory subunits being located on opposite sides of the membrane in each system, it is clear that each system is functionally equivalent with bacterial proteins having the ability to use the eukaryotic SAM and vice versa. In this review, we summarize the similarities and differences between the BAM and SAM complexes, highlighting the possible selecting pressures on bacteria and eukaryotes during evolution. It is also now emerging that bacterial pathogens utilize the SAM to target toxins and effector proteins to host mitochondria and this will also be discussed from an evolutionary perspective.
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Int. J. Mol. Sci. 2012, 13, 8038-8050; doi:10.3390/ijms13078038
International Journal of
Molecular Sciences
ISSN 1422-0067
www.mdpi.com/journal/ijms
Review
From Evolution to Pathogenesis: The Link Between β-Barrel
Assembly Machineries in the Outer Membrane of Mitochondria
and Gram-Negative Bacteria
Jhih-Hang Jiang, Janette Tong, Kher Shing Tan and Kipros Gabriel *
Department of Biochemistry and Molecular Biology, Monash University, Clayton Campus,
Melbourne 3800, Australia; E-Mails: jhih-hang.jiang@monash.edu (J.-H.J.);
janette.tong@monash.edu (J.T.); kher.tan@monash.edu (K.S.T.)
* Author to whom correspondence should be addressed; E-Mail: kip.gabriel@monash.edu;
Tel.: +61-3-9902-9213; Fax: +61-3-9905-3726.
Received: 1 May 2012; in revised form: 21 June 2012 / Accepted: 21 June 2012 /
Published: 28 June 2012
Abstract: β-barrel proteins are the highly abundant in the outer membranes of
Gram-negative bacteria and the mitochondria in eukaryotes. The assembly of β-barrels is
mediated by two evolutionary conserved machineries; the β-barrel Assembly Machinery
(BAM) in Gram-negative bacteria; and the Sorting and Assembly Machinery (SAM) in
mitochondria. Although the BAM and SAM have functionally conserved roles in the
membrane integration and folding of β-barrel proteins, apart from the central BamA
and Sam50 proteins, the remaining components of each of the complexes have diverged
remarkably. For example all of the accessory components of the BAM complex characterized
to date are located in the bacterial periplasm, on the same side as the N-terminal domain of
BamA. This is the same side of the membrane as the substrates that are delivered to the
BAM. On the other hand, all of the accessory components of the SAM complex are located
on the cytosolic side of the membrane, the opposite side of the membrane to the
N-terminus of Sam50 and the substrate receiving side of the membrane. Despite the
accessory subunits being located on opposite sides of the membrane in each system, it is
clear that each system is functionally equivalent with bacterial proteins having the ability
to use the eukaryotic SAM and vice versa. In this review, we summarize the similarities
and differences between the BAM and SAM complexes, highlighting the possible selecting
pressures on bacteria and eukaryotes during evolution. It is also now emerging that
OPEN ACCESS
Int. J. Mol. Sci. 2012, 13 8039
bacterial pathogens utilize the SAM to target toxins and effector proteins to host
mitochondria and this will also be discussed from an evolutionary perspective.
Keywords: β-barrel proteins; mitochondria; bacteria; outer membrane; protein transport;
protein folding; evolution
1. Basic Features of the Bacterial and Mitochondrial Membranes
Gram-negative bacteria contain two lipid bilayers which envelope the periplasm: the inner
membrane (IM) and outer membrane (OM). The asymmetric outer membrane is composed of
lipopolysaccharides (LPS) in the outer leaflet and phospholipid in the inner leaflet while the symmetric
inner membrane is composed of a phospholipid bilayer. The outer membrane predominantly harbors
β-barrel fold proteins whereas α-helical based transmembrane proteins are primarily found in the inner
membrane. The outer membrane provides the physical barrier from the outside world, but also must
maintain selective permeability for the uptake of nutrients to support physiological functions, for the
release of virulence factors and in some cases for multidrug resistance. This is achieved by the outer
membrane proteins [1,2]. Evolutionarily, mitochondria are derived from an ancient α-proteobacterium
through an endosymbiotic relationship that was initiated billions of years ago [3–5]. They have
retained their double membrane structure that forms the inner and outer membranes, which enclose
two aqueous environments, the intermembrane space and the matrix. This allows for the separation
of biochemical processes and hence mitochondria perform many roles in eukaryotic cells. To carry
out these functions, nutrients, biochemical intermediates, lipids and proteins must traverse the
mitochondrial membranes. Outer membrane proteins (OMPs), particularly β-barrel proteins, play a key
role in these transport processes [6].
2. β-Barrel Proteins and the Assembly in the Bacterial and Mitochondrial Outer Membrane
β-barrel proteins are defined as anti-parallel β-strands forming larger β-sheets that are connected
through hydrogen bonds between the transmembrane strands that confer extreme stability [7]. As most
β-barrels are membrane spanning proteins, it is typical for them to possess amino acids with
hydrophobic side chains on their surface that abuts the phospholipids, while hydrophilic or charged
residues often face the central cavity [1,7,8].
The assembly of β-barrel OMPs is mediated by sophisticated molecular machineries.
In Gram-negative bacteria, nascent polypeptides are synthesized with an amino-terminal signal peptide
by cytoplasmic ribosomes. These precursors are bound by factors such as SecB and SecA or the
chaperone DnaK and transported to the secretion machinery (Sec). The Sec translocates proteins into
the periplasm. Once in the periplasm, the signal peptide is processed, and precursor proteins associate
with the chaperones SurA and Skp which not only transfer proteins to the BAM complex for outer
membrane insertion but also prevent their aggregation [9]. A recent study has also shown that
chaperones also play an important role in assisting folding and assembly of β-barrel precursor proteins,
using in vitro reconstitution techniques with BAM and its substrates [10].
Int. J. Mol. Sci. 2012, 13 8040
Similarly, in mitochondria, precursor β-barrel proteins are also synthesized by cytosolic ribosomes
and are bound by chaperones in the cytosol [11]. These precursor substrates are transported to the
receptor subunits of the Translocase of the Outer Mitochondrial membrane (TOM). The TOM complex
translocates substrate proteins across the outer membrane into the intermembrane space. The small
chaperones Tim9/10 or Tim8/13 associate with substrates, in a manner which is functionally analogous
to that performed by SurA and Skp in bacteria, for the transfer to the Sorting and Assembly Machinery
(SAM) complex at the mitochondrial outer membrane [12–17].
3. The β-Barrel Assembly Machinery in Gram-Negative Bacteria
In bacteria, the Omp85 family of protein assembly machines is the key and essential component of
outer membrane β-barrel protein insertion processes. Omp85 protein homologues are found in bacteria
and eukaryotes, including fungi, plants (mitochondria and chloroplasts) and animals [15–19]. Study of
the BamA protein in N. meningitidis [18] and Escherichia coli [20–22] first revealed the essential role
of Omp85 protein machines in folding and assembly of outer membrane proteins. BamA is highly
conserved throughout all Gram-negative bacterial species [18]. Structural predictions relating to BamA
suggest that it is composed of two parts: a large soluble N-terminal domain protruding to the periplasm;
and the carboxy-terminal β-barrel domain sitting in the OM. The large soluble domain has five repeat
POTRA (polypeptide transport-associated) domain, these repeat domains are suggested to bind
unfolded β-barrel proteins [23]. The deletion of each POTRA domain exerts different effects to
complex stability and substrate binding. POTRA domains 2 to 4 are important for the interaction with
the partner protein in the BAM complex, BamB, whereas deletion of POTRA 5 specifically affects
association with other lipoprotein partners [24,25].
Defects in BAM complex function have many deleterious primary and secondary effects, including
accumulation of unfolded protein substrates and reduced LPS and phospholipid incorporation into the
outer membrane. Failures in these processes would cause accumulation of these factors at the inner
membrane [26]. Recent evidence indicates BamA acts as the assembly hub for the formation of an
LptD/E complex in the outer membrane, which is subsequently essential for LPS biogenesis [27].
In E. coli, BamA (previously referred as YaeT) is associated with four lipoproteins BamB, C, D and E
(previously known as YfgL, NlpB, YfiO and SmpA respectively) [20,28–30]. The BAM is also
involved in autotransporter protein biogenesis, which is not surprising as autotransporters have a
C-terminal β-barrel domain [31,32]. The N-terminus of autotransporter proteins consist of passenger
domains which are translocated through the folded barrel formed by the C-terminus and usually act as
virulence factors during infection. The BAM was shown to be associated with stalled autotransporter
substrates created by an in vivo site-specific photocrosslinking approach. It is hypothesized that the
β-barrel domain initially interacts with chaperones in the periplasm and then passed to BamA, BamB
and BamD sequentially. As both BamB and BamD are still found crosslinked to substrates at later time
points during chase experiments, it has been suggested that they are involved in the late stages of
folding/assembly. The passenger domain of autotransporter EspP can transiently interact with the
channel BamA and the release of passenger domain seems to be a checkpoint for the completion of
assembly of β-barrel domain [31,32].
Int. J. Mol. Sci. 2012, 13 8041
4. The Sorting and Assembly Machinery (SAM) in Mitochondria
The key molecule of the eukaryotic SAM is the β-barrel core protein, Sam50, the homolog of the
bacterial protein BamA. Sam50 is predicted to have one POTRA domain that faces the mitochondrial
intermembrane space, the equivalent of the bacterial periplasm. Thus, assembly of β-barrels occurs
from the same face of the membrane in bacteria and mitochondria as substrates are first imported by
the TOM complex into the intermembrane space before they engage with the SAM. This suggests that
the functional features of the eukaryotic system have been retained from the endosymbiont [33,34].
The POTRA domain of Sam50 was once reported as a signal receptor for β-barrel protein as deletion
of a short segment caused a severe phenotype and a loss in SAM-substrate interaction [35]. The role of
POTRA in Sam50 is now thought to facilitate the folded β-barrel release into the mitochondrial outer
membrane instead of having a signal receptor role as deletion of the entire POTRA domain results
in little change from the wild type phenotype and does not affect the kinetics of β-barrel assembly
in vitro [36,37].
In addition to the central protein, Sam50, the SAM complex is also composed of two peripheral
subunits that face the cytosol, Sam35 and Sam37. Two other integral membrane proteins also
transiently act in the β-barrel folding pathway, Mdm10 and Mim1 [11,38,39]. Only Sam50 is
evolutionarily conserved from Gram-negative bacteria to mitochondria (Figure 1) [15–17]. Moreover
the peripheral subunits, Sam35 and Sam37, are actually located on the opposite side of the membrane
to the intermembrane space and hence the POTRA domain. This is in contrast to the bacterial system
where BamB to E have large domains in the periplasm. Sam35 is a receptor that binds the β-signal of
the substrate β-barrel proteins, stabilizing the substrate proteins in the SAM complex whilst strands are
being inserted into the membrane [37,40]. Sam37 promotes the release of β-barrel substrate proteins
from SAM complex at a later stage of folding [40]. The SAM complex is not only considered to be a
β-barrel protein folding station. It is also associated with the post-folding transfer and incorporation of
proteins into their respective native complexes; it also plays a role in connections and communication
with other organelles. Hence it is considered a “hub” for membrane protein biogenesis and organelle
communication [41–43]. The metaxin1 and 2 proteins in mammalian cells are considered to perform
similar roles to Sam35 and Sam37 [44].
Critically, the SAM complex serves as the initial platform for assembly of the TOM complex that is
the entry portal for all imported mitochondrial proteins. The central component of the TOM complex,
the protein conducting pore, Tom40, is also a β-barrel protein. Tom40 needs to be assembled with a
series of receptor subunits including, Tom20, Tom22 and Tom70, as well as three small Tom proteins
(Tom5, Tom6, and Tom7) that seem to have a complex organising role [38,45–48].
Two other proteins which form transient associations with the SAM are Mdm10 and
Mdm12 [38,39,49]. Collectively these proteins are also important for the formation of the
endoplasmic-reticulum mitochondria encounter structure (ERMES) [38,39,41,42,49–53]. Mim1 also
interacts with the SAM complex and is important for the biogenesis of outer membrane tethered
α-helical transmembrane domain proteins [54,55].
A recent report also implicates Sam50 in the maintenance of cristae structure and respiration by
serving as a bridging point between the outer and inner mitochondrial membrane [56]. This emphasizes the
multi-functional role of the SAM in mitochondrial biogenesis, function and maintenance. These
Int. J. Mol. Sci. 2012, 13 8042
multiple roles can be explained by the evolutionary view that eukaryotic cells developed new
mitochondrial functions based on pre-existing protein molecules as scaffolds as well as keeping the
original functions of these molecules. The role of Sam50 as a β-barrel protein insertion and folding
machine in addition to the many other roles of the SAM complex are testament to this theory.
Figure 1. Comparison of the β-barrel protein assembly machineries in Gram-negative
bacteria and eukaryotes (A) In Gram-negative bacteria, BamA is the core subunit of the
BAM complex. BamA forms a complex with lipoproteins, BamB, C, D and E in E. coli.
The BAM complex plays an important role in outer membrane biogenesis including the
insertion of autotransporters; (B) In eukaryotes, the core channel of SAM, Sam50, is the
homologue of BamA. In mitochondria, Sam50 forms a complex with the metaxins, Sam35
and Sam37. Mdm10 interacts with SAM to mediate the biogenesis of TOM. Mim1
transiently interacts with the SAM complex and is important for the biogenesis of α-helical
transmembrane domain containing proteins. BamA has five repeat POTRA domains
(P1–P5) while Sam50 only has one (P1). In eukaryotes the accessory subunits are located
on the cytosolic side of the outer membrane whilst in bacteria BamB to E are located on
the periplasmic face of the membrane. Color key-grey denotes components not conserved
between prokaryotes and eukaryotes, blue denotes conserved proteins.
5. How Functionally Conserved Are the BAM and SAM?
Since the discovery of both BamA and Sam50, many bacterial β-barrel proteins have been used to
test the ability of SAM complex to fold bacterial β-barrel proteins in mitochondria [57–60] (Table 1).
The 8-stranded monomeric barrel OmpA, the 16-stranded OmpC and PhoE from of E. coli, and
Omp85 from Neisseria meningitidis have been reported to target to mitochondria when they were
expressed in the eukaryote Saccharomyces cerevisiae. Sam50 and Sam37 were shown to be required
for PhoE assembly using genetically modified yeast strains [57]. The C-terminal residue of PhoE,
which is required for the assembly in bacteria, is also suggested to be important for the trimeric
Int. J. Mol. Sci. 2012, 13 8043
formation of PhoE in mitochondria [57,61]. On the other hand, expression of OmpA, OmpC or PhoE
in mammalian cell lines fails to result in successful targeting and assembly in mitochondria. The
Omp85 and PorB proteins from Neisseria gonorrhoeae are recognized and inserted and folded into the
mitochondrial outer membrane by the SAM in mammalian and yeast derived mitochondria [58,60].
Furthermore, expression of a different class of bacterial β-barrel, where more than one protein subunit
is required for complete barrel formation, was also shown to be possible in mitochondria. The barrel
domain of the YadA autotransporter from Yersinia enterocolitica expressed in the yeast S. cerevisiae
showed that eukaryotes could assemble these fragments into a barrel. Trimeric YadA assembles as a
12-stranded β-barrel with a contribution of 4-strands from each YadA. The assembly process requires
the intermembrane space chaperone proteins and the SAM complex [59]. The reasons why some and
not other bacterial proteins can be imported and folded in eukaryotic mitochondria are not known but
is likely to be related to the presence or absence of signal sequences that target them to mitochondria
and secondly be able to be recognized by the SAM.
Table 1 . Tests of bacterial β-barrel assembly in mitochondria.
Bacterial species β-barrel protein References
Bacterial β-barrels target to mitochondria during infection
Neisseria gonorrhoeae PorB [58,60,62–64]
Neisseria meningitidis PorB [60,65,66]
Acinetobacter baumannii Omp38 (AbOmpA) [67]
Staphylococcus aureus Panton-Valentine leukocidin (PVL) [68]
Bacterial β-barrels tested to be expressed in mitochondria
E. coli PhoE, OmpA, OmpC [57,58]
Yersinia enterocolitica Ya d A [59]
Neisseria meningitidis Omp85(BamA) [57]
Neisseria gonorrhoeae Omp85(BamA) [58]
Reciprocal experiments examining the folding and assembly of eukaryotic β-barrel proteins in
bacteria have also shown that bacterial machineries can assemble and fold eukaryotic β-barrel proteins
in the bacterial outer membrane. The eukaryotic mitochondrial porin, from Neurospora crassa, one of
the most abundant β-barrel outer mitochondrial membrane proteins of eukaryotes, can be assembled
into the bacterial outer membrane [69].
6. β-Barrels—Evolution and Pathogenesis
Mitochondria play dominant roles in cell death and energy metabolism [70]. During bacterial
infection, many effector proteins are transferred to host cells for the manipulation of cell function [71].
It is therefore not surprising that mitochondria are targets of bacterial toxins [72]. The fact that
bacterial β-barrels can in fact be assembled in mitochondria is not only intriguing from an evolutionary
perspective, but it is now becoming clearer. Evolutionary conserved principles are used by some
bacteria to target β-barrel toxins to host mitochondrial outer membranes via the SAM.
The bacterial outer membrane protein PorB from Neisseria gonorrhoeae (gonococcal) and
Neisseria meningitidis (meningococcal) have been found to be targeted to the mitochondria during
infection [58,60,62–66]. PorB is a β-barrel protein composed of 16 β-strands and has similar
Int. J. Mol. Sci. 2012, 13 8044
electrophysiological activity as eukaryotic porins that can be regulated by purine triphosphate
nucleotides [73,74]. The localization of PorB in mitochondria was once controversial. Meningococcal
PorB was suggested to target to the mitochondrial outer membrane while gonococcal PorB was once
reported to randomly insert into mitochondrial inner membrane [62,65]. The exact localization of PorB
in mitochondria was recently examined using a novel method combining in vitro mitochondrial import
assays and β-barrel mobility gel shift assays. PorB assembles in the mitochondrial outer membrane
using the SAM core subunit Sam50 [60]. The molecular mechanisms behind PorB targeting to
mitochondria can be attributed to the evolutionary link between mitochondria and their bacterial
ancestors. PorB enters mitochondria through the TOM complex like all endogenous eukaryotic
β-barrels [64]. After entry into mitochondria, the chaperone proteins in the intermembrane space, the
small Tims, are required for transport of PorB to the SAM in the outer membrane [60]. Interestingly,
the accessory subunits of the SAM, Sam35 and Sam37, which are important for eukaryotic β-barrel
folding, are not required for PorB assembly in mitochondria [60]. Only the core SAM subunit, Sam50,
is required. Furthermore, unlike eukaryotic β-barrels, PorB does not have a strict eukaryotic like
β-signal at the carboxy-terminus [37,60]. This could be explained by the fact that β-signal regions that
evolved in mitochondria and bacteria have diverged, with the signal in eukaryotes co-evolving with
Sam35 and Sam37. The signal regions in eukaryotes could have a greater role in the regulation of the
rate of folding but may not be absolutely required and hence are not found universally in bacteria.
Although it is generally agreed that PorB can localize to the mitochondria, its exact function remains
controversial with both pro and anti-apoptotic effects reported [62,66].
Another pathogenic β-barrel Omp38 (AbOmpA) from Acinetobacter baumannii has also been
found to target mitochondria and reported to promote cell death [67]. AbOmpA is a bacterial outer
membrane protein which has a C-terminal OmpA-like domain which can bind peptidoglycan [75]. The
localization of AbOmpA in mitochondria and the molecular mechanisms behind import are still
unclear but it would be anticipated that it may also use the SAM for its membrane folding/insertion.
In contrast to traditionally studied β-barrel proteins, other β-stranded bacterial proteins that are not
likely to use the SAM also exist. Panton-Valentine leukocidin (PVL) from Staphylococcus aureus has
also been identified as a mitochondrial targeted that induces apoptosis in neutrophils [68]. PVL is a
bi-component toxin composed of two subunits, LukF-PV and LukS-PV, with the ability to form
β-barrel in membranes [76,77]. However, ability of PVL to form pores at either the mitochondrial inner
or outer membrane is yet to be demonstrated. Structural information also suggests that PVL is unlikely to
utilize the SAM as it is predicted to be structurally and functionally analogous to other leukocidin and
γ-haemolysin like toxins that firstly form multimers and a channel like structure at the membrane
surface before they insert into the membrane without assistance from other machineries [78].
7. Concluding Remarks and Future Directions
Hosts and pathogens co-evolve, with advantageous traits of hosts or pathogens selected for in a
given population. For bacterial pathogens this may mean that for example; a surface factor is altered to
better evade host immune defences; or a modified and more potent toxin is produced. More potent
toxins could be the result of a mutational change that alters the way a toxin interacts with its host target
protein or pathway, but it may also be the result of changing the efficiency of transport of the toxin to
Int. J. Mol. Sci. 2012, 13 8045
its target site. We propose that some pathogens, through selective forces, have continued to produce
β-barrel toxins that can be transported to mitochondria with high efficiency. As mitochondria have
diverged from their bacterial ancestors and adapted their mechanism for the import/assembly of
β-barrel outer membrane proteins to their cellular context, the bacterial pathogens that produce
β-barrel toxins that are targeted to mitochondria have by necessity also changed. Any changes in the
way the SAM recognizes β-barrel proteins, in particular if this results in changes in any signal regions
of the β-barrel substrate proteins, need to be countered by the bacterial pathogens. It will be interesting
to compare the efficiencies of import and assembly of β-barrel toxins into mammalian mitochondria
with non-toxin bacterial β-barrels on a large scale to assess for the differences as our list of bacterial
β-barrel toxins increases.
Research in the area of β-barrel protein biogenesis in Gram-negative bacteria and mitochondria
during the past decade highlights the link between the two β-barrel assembly machineries, the BAM
and SAM, respectively. The last ten years of research have revealed much about these remarkable
machines, though future research will elucidate many of the mechanistic details that are still unclear.
It is now emerging that due to their evolutionary heritage, mitochondria are targets of bacterial β-barrel
toxins. As more β-barrel bacterial toxins are discovered, the battery of substrates to probe SAM
function will increase, hopefully allowing us to understand how the exact mechanisms utilised by
BAM and SAM to insert and fold proteins in the membrane in addition to better understanding
mechanisms at play during infection.
References
1. Koebnik, R.; Locher, K.P.; van Gelder, P. Structure and function of bacterial outer membrane
proteins: Barrels in a nutshell. Mol. Microbiol. 2000, 37, 239–253.
2. Bos, M.P.; Robert, V.; Tommassen, J. Biogenesis of the gram-negative bacterial outer membrane.
Annu. Rev. Microbiol. 2007, 61, 191–214.
3. Gray, M.W.; Burger, G.; Lang, B.F. Mitochondrial evolution. Science 1999, 283, 1476–1481.
4. Yang, D.; Oyaizu, Y.; Oyaizu, H.; Olsen, G.J.; Woese, C.R. Mitochondrial origins. Proc. Natl.
Acad. Sci. USA 1985, 82, 4443–4447.
5. Gray, M.W. The incredible shrinking organelle. EMBO Rep. 2011, 12, doi:10.1038/embor.2011.168.
6. Endo, T.; Yamano, K. Transport of proteins across or into the mitochondrial outer membrane.
Biochim. Biophys. Acta 2010, 1803, 706–714.
7. Fairman, J.W.; Noinaj, N.; Buchanan, S.K. The structural biology of β-barrel membrane proteins:
A summary of recent reports. Curr. Opin. Struct. Biol. 2011, 21, 523–531.
8. Burgess, N.K.; Dao, T.P.; Stanley, A.M.; Fleming, K.G. β-barrel proteins that reside in the
Escherichia coli outer membrane in vivo demonstrate varied folding behavior in vitro. J. Biol.
Chem. 2008, 283, 26748–26758.
9. Hagan, C.L.; Silhavy, T.J.; Kahne, D. β-Barrel membrane protein assembly by the Bam complex.
Annu. Rev. Biochem. 2011, 80, 189–210.
10. Hagan, C.L.; Kim, S.; Kahne, D. Reconstitution of outer membrane protein assembly from purified
components. Science 2010, 328, 890–892.
Int. J. Mol. Sci. 2012, 13 8046
11. Chacinska, A.; Koehler, C.M.; Milenkovic, D.; Lithgow, T.; Pfanner, N. Importing mitochondrial
proteins: Machineries and mechanisms. Cell 2009, 138, 628–644.
12. Hoppins, S.C.; Nargang, F.E. The Tim8-Tim13 complex of Neurospora crassa functions in the
assembly of proteins into both mitochondrial membranes. J. Biol. Chem. 2004, 279, 12396–12405.
13. Wiedemann, N.; Truscott, K.N.; Pfannschmidt, S.; Guiard, B.; Meisinger, C.; Pfanner, N.
Biogenesis of the protein import channel Tom40 of the mitochondrial outer membrane:
Intermembrane space components are involved in an early stage of the assembly pathway. J. Biol.
Chem. 2004, 279, 18188–18194.
14. Wiedemann, N.; Kozjak, V.; Chacinska, A.; Schonfisch, B.; Rospert, S.; Ryan, M.T.; Pfanner, N.;
Meisinger, C. Machinery for protein sorting and assembly in the mitochondrial outer membrane.
Nature 2003, 424, 565–571.
15. Paschen, S.A.; Waizenegger, T.; Stan, T.; Preuss, M.; Cyrklaff, M.; Hell, K.; Rapaport, D.;
Neupert, W. Evolutionary conservation of biogenesis of beta-barrel membrane proteins. Nature
2003, 426, 862–866.
16. Gentle, I.; Gabriel, K.; Beech, P.; Waller, R.; Lithgow, T. The Omp85 family of proteins is essential
for outer membrane biogenesis in mitochondria and bacteria. J. Cell Biol. 2004, 164, 19–24.
17. Kozjak, V.; Wiedemann, N.; Milenkovic, D.; Lohaus, C.; Meyer, H.E.; Guiard, B.; Meisinger, C.;
Pfanner, N. An essential role of Sam50 in the protein sorting and assembly machinery of the
mitochondrial outer membrane. J. Biol. Chem. 2003, 278, 48520–48523.
18. Voulhoux, R.; Bos, M.P.; Geurtsen, J.; Mols, M.; Tommassen, J. Role of a highly conserved
bacterial protein in outer membrane protein assembly. Science 2003, 299, 262–265.
19. Reumann, S.; Davila-Aponte, J.; Keegstra, K. The evolutionary origin of the protein-translocating
channel of chloroplastic envelope membranes: Identification of a cyanobacterial homolog.
Proc. Natl. Acad. Sci. USA 1999, 96, 784–789.
20. Wu, T.; Malinverni, J.; Ruiz, N.; Kim, S.; Silhavy, T.J.; Kahne, D. Identification of a
multicomponent complex required for outer membrane biogenesis in Escherichia coli. Cell 2005,
121, 235–245.
21. Werner, J.; Misra, R. YaeT (Omp85) affects the assembly of lipid-dependent and lipid-independent
outer membrane proteins of Escherichia coli. Mol. Microbiol. 2005, 57, 1450–1459.
22. Doerrler, W.T.; Raetz, C.R. Loss of outer membrane proteins without inhibition of lipid export in
an Escherichia coli YaeT mutant. J. Biol. Chem. 2005, 280, 27679–27687.
23. Sanchez-Pulido, L.; Devos, D.; Genevrois, S.; Vicente, M.; Valencia, A. POTRA: A conserved
domain in the FtsQ family and a class of beta-barrel outer membrane proteins. Trends Biochem. Sci.
2003, 28, 523–526.
24. Kim, S.; Malinverni, J.C.; Sliz, P.; Silhavy, T.J.; Harrison, S.C.; Kahne, D. Structure and function
of an essential component of the outer membrane protein assembly machine. Science 2007, 317,
961–964.
25. Bos, M.P.; Robert, V.; Tommassen, J. Functioning of outer membrane protein assembly factor
Omp85 requires a single POTRA domain. EMBO Rep. 2007, 8, 1149–1154.
26. Genevrois, S.; Steeghs, L.; Roholl, P.; Letesson, J.J.; van der Ley, P. The Omp85 protein of
Neisseria meningitidis is required for lipid export to the outer membrane. EMBO J. 2003, 22,
1780–1789.
Int. J. Mol. Sci. 2012, 13 8047
27. Chimalakonda, G.; Ruiz, N.; Chng, S.S.; Garner, R.A.; Kahne, D.; Silhavy, T.J. Lipoprotein LptE is
required for the assembly of LptD by the beta-barrel assembly machine in the outer membrane of
Escherichia coli. Proc. Natl. Acad. Sci. USA 2011, 108, 2492–2497.
28. Malinverni, J.C.; Werner, J.; Kim, S.; Sklar, J.G.; Kahne, D.; Misra, R.; Silhavy, T.J. YfiO
stabilizes the YaeT complex and is essential for outer membrane protein assembly in
Escherichia coli. Mol. Microbiol. 2006, 61, 151–164.
29. Sklar, J.G.; Wu, T.; Gronenberg, L.S.; Malinverni, J.C.; Kahne, D.; Silhavy, T.J. Lipoprotein SmpA
is a component of the YaeT complex that assembles outer membrane proteins in Escherichia coli.
Proc. Natl. Acad. Sci. USA 2007, 104, 6400–6405.
30. Hagan, C.L.; Silhavy, T.J.; Kahne, D.E. β-Barrel Membrane Protein Assembly by the Bam
Complex. Annu. Rev. Biochem. 2011, 80, 189–210.
31. Ieva, R.; Bernstein, H.D. Interaction of an autotransporter passenger domain with BamA during
its translocation across the bacterial outer membrane. Proc. Natl. Acad. Sci. USA 2009, 106,
19120–19125.
32. Ieva, R.; Tian, P.; Peterson, J.H.; Bernstein, H.D. Sequential and spatially restricted interactions
of assembly factors with an autotransporter beta domain. Proc. Natl. Acad. Sci. USA 2011, 108,
E383–E391.
33. Walther, D.M.; Rapaport, D.; Tommassen, J. Biogenesis of β-barrel membrane proteins in bacteria
and eukaryotes: Evolutionary conservation and divergence. Cell. Mol. Life Sci. 2009, 66,
2789–2804.
34. Gatsos, X.; Perry, A.J.; Anwari, K.; Dolezal, P.; Wolynec, P.P.; Likic, V.A.; Purcell, A.W.;
Buchanan, S.K.; Lithgow, T. Protein secretion and outer membrane assembly in
Alphaproteobacteria. FEMS Microbiol. Rev. 2008, 32, 995–1009.
35. Habib, S.J.; Waizenegger, T.; Niewienda, A.; Paschen, S.A.; Neupert, W.; Rapaport, D. The
N-terminal domain of Tob55 has a receptor-like function in the biogenesis of mitochondrial
β-barrel proteins. J. Cell Biol. 2007, 176, 77–88.
36. Stroud, D.A.; Becker, T.; Qiu, J.; Stojanovski, D.; Pfannschmidt, S.; Wirth, C.; Hunte, C.;
Guiard, B.; Meisinger, C.; Pfanner, N.; et al. Biogenesis of mitochondrial beta-barrel proteins: The
POTRA domain is involved in precursor release from the SAM complex. Mol. Biol. Cell 2011, 22,
2823–2833.
37. Kutik, S.; Stojanovski, D.; Becker, L.; Becker, T.; Meinecke, M.; Kruger, V.; Prinz, C.; Meisinger, C.;
Guiard, B.; Wagner, R.; et al. Dissecting membrane insertion of mitochondrial β-barrel proteins.
Cell 2008, 132, 1011–1024.
38. Meisinger, C.; Rissler, M.; Chacinska, A.; Szklarz, L.K.; Milenkovic, D.; Kozjak, V.; Schonfisch, B.;
Lohaus, C.; Meyer, H.E.; Yaffe, M.P.; et al. The mitochondrial morphology protein Mdm10
functions in assembly of the preprotein translocase of the outer membrane. Dev. Cell 2004, 7,
61–71.
39. Meisinger, C.; Pfannschmidt, S.; Rissler, M.; Milenkovic, D.; Becker, T.; Stojanovski, D.;
Youngman, M.J.; Jensen, R.E.; Chacinska, A.; Guiard, B.; et al. The morphology proteins
Mdm12/Mmm1 function in the major beta-barrel assembly pathway of mitochondria. EMBO J.
2007, 26, 2229–2239.
Int. J. Mol. Sci. 2012, 13 8048
40. Chan, N.C.; Lithgow, T. The peripheral membrane subunits of the SAM complex function
codependently in mitochondrial outer membrane biogenesis. Mol. Biol. Cell 2008, 19, 126–136.
41. Kornmann, B.; Currie, E.; Collins, S.R.; Schuldiner, M.; Nunnari, J.; Weissman, J.S.; Walter, P.
An ER-mitochondria tethering complex revealed by a synthetic biology screen. Science 2009, 325,
477–481.
42. Thornton, N.; Stroud, D.A.; Milenkovic, D.; Guiard, B.; Pfanner, N.; Becker, T. Two modular
forms of the mitochondrial sorting and assembly machinery are involved in biogenesis of α-helical
outer membrane proteins. J. Mol. Biol. 2010, 396, 540–549.
43. Wiedemann, N.; Meisinger, C.; Pfanner, N. Cell biology. Connecting organelles. Science 2009,
325, 403–404.
44. Kozjak-Pavlovic, V.; Ross, K.; Benlasfer, N.; Kimmig, S.; Karlas, A.; Rudel, T. Conserved roles of
Sam50 and metaxins in VDAC biogenesis. EMBO Rep. 2007, 8, 576–582.
45. Hill, K.; Model, K.; Ryan, M.T.; Dietmeier, K.; Martin, F.; Wagner, R.; Pfanner, N. Tom40 forms
the hydrophilic channel of the mitochondrial import pore for preproteins [see comment]. Nature
1998, 395, 516–521.
46. Meisinger, C.; Ryan, M.T.; Hill, K.; Model, K.; Lim, J.H.; Sickmann, A.; Muller, H.; Meyer, H.E.;
Wagner, R.; Pfanner, N. Protein import channel of the outer mitochondrial membrane: A highly
stable Tom40-Tom22 core structure differentially interacts with preproteins, small tom proteins,
and import receptors. Mol. Cell. Biol. 2001, 21, 2337–2348.
47. Model, K.; Meisinger, C.; Prinz, T.; Wiedemann, N.; Truscott, K.N.; Pfanner, N.; Ryan, M.T.
Multistep assembly of the protein import channel of the mitochondrial outer membrane. Nat. Struct.
Biol. 2001, 8, 361–370.
48. Model, K.; Prinz, T.; Ruiz, T.; Radermacher, M.; Krimmer, T.; Kuhlbrandt, W.; Pfanner, N.;
Meisinger, C. Protein translocase of the outer mitochondrial membrane: Role of import receptors in
the structural organization of the TOM complex. J. Mol. Biol. 2002, 316, 657–666.
49. Wideman, J.G.; Go, N.E.; Klein, A.; Redmond, E.; Lackey, S.W.; Tao, T.; Kalbacher, H.; Rapaport, D.;
Neupert, W.; Nargang, F.E. Roles of the Mdm10, Tom7, Mdm12, and Mmm1 proteins in the
assembly of mitochondrial outer membrane proteins in Neurospora crassa. Mol. Biol. Cell 2010, 21,
1725–1736.
50. Becker, T.; Wenz, L.S.; Thornton, N.; Stroud, D.; Meisinger, C.; Wiedemann, N.; Pfanner, N.
Biogenesis of mitochondria: Dual role of Tom7 in modulating assembly of the preprotein
translocase of the outer membrane. J. Mol. Biol. 2011, 405, 113–124.
51. Yamano, K.; Tanaka-Yamano, S.; Endo, T. Mdm10 as a dynamic constituent of the TOB/SAM
complex directs coordinated assembly of Tom40. EMBO Rep. 2010, 11, 187–193.
52. Yamano, K.; Tanaka-Yamano, S.; Endo, T. Tom7 regulates Mdm10-mediated assembly of the
mitochondrial import channel protein Tom40. J. Biol. Chem. 2010, 285, 41222–41231.
53. Kornmann, B. ERMES, a multifunctional complex connecting endoplasmic reticulum and
mitochondria. Med. Sci. (Paris) 2010, 26, 145–146.
54. Becker, T.; Pfannschmidt, S.; Guiard, B.; Stojanovski, D.; Milenkovic, D.; Kutik, S.; Pfanner, N.;
Meisinger, C.; Wiedemann, N. Biogenesis of the mitochondrial TOM complex: Mim1 promotes
insertion and assembly of signal-anchored receptors. J. Biol. Chem. 2008, 283, 120–127.
Int. J. Mol. Sci. 2012, 13 8049
55. Hulett, J.M.; Lueder, F.; Chan, N.C.; Perry, A.J.; Wolynec, P.; Likic, V.A.; Gooley, P.R.; Lithgow, T.
The transmembrane segment of Tom20 is recognized by Mim1 for docking to the mitochondrial
TOM complex. J. Mol. Biol. 2008, 376, 694–704.
56. Ott, C.; Ross, K.; Straub, S.; Thiede, B.; Gotz, M.; Goosmann, C.; Krischke, M.; Mueller, M.J.;
Krohne, G.; Rudel, T.; et al. Sam50 functions in mitochondrial intermembrane space bridging and
biogenesis of respiratory complexes. Mol. Cell. Biol. 2012, 32, 1173–1188.
57. Walther, D.M.; Papic, D.; Bos, M.P.; Tommassen, J.; Rapaport, D. Signals in bacterial β-barrel
proteins are functional in eukaryotic cells for targeting to and assembly in mitochondria. Proc. Natl.
Acad. Sci. USA 2009, 106, 2531–2536.
58. Kozjak-Pavlovic, V.; Ott, C.; Gotz, M.; Rudel, T. Neisserial Omp85 protein is selectively
recognized and assembled into functional complexes in the outer membrane of human
mitochondria. J. Biol. Chem. 2011, 286, 27019–27026.
59. Muller, J.E.; Papic, D.; Ulrich, T.; Grin, I.; Schutz, M.; Oberhettinger, P.; Tommassen, J.;
Linke, D.; Dimmer, K.S.; Autenrieth, I.B.; et al. Mitochondria can recognize and assemble
fragments of a beta-barrel structure. Mol. Biol. Cell 2011, 22, 1638–1647.
60. Jiang, J.H.; Davies, J.K.; Lithgow, T.; Strugnell, R.A.; Gabriel, K. Targeting of Neisserial PorB to
the mitochondrial outer membrane: An insight on the evolution of β-barrel protein assembly
machines. Mol. Microbiol. 2011, 82, 976–987.
61. Struyve, M.; Moons, M.; Tommassen, J. Carboxy-terminal phenylalanine is essential for the correct
assembly of a bacterial outer membrane protein. J. Mol. Biol. 1991, 218, 141–148.
62. Kozjak-Pavlovic, V.; Dian-Lothrop, E.A.; Meinecke, M.; Kepp, O.; Ross, K.; Rajalingam, K.;
Harsman, A.; Hauf, E.; Brinkmann, V.; Gunther, D.; et al. Bacterial porin disrupts mitochondrial
membrane potential and sensitizes host cells to apoptosis. PLoS Pathog. 2009, 5,
doi:10.1371/journal.ppat.1000629.
63. Muller, A.; Gunther, D.; Brinkmann, V.; Hurwitz, R.; Meyer, T.F.; Rudel, T. Targeting of the
pro-apoptotic VDAC-like porin (PorB) of Neisseria gonorrhoeae to mitochondria of infected cells.
EMBO J. 2000, 19, 5332–5343.
64. Muller, A.; Rassow, J.; Grimm, J.; Machuy, N.; Meyer, T.F.; Rudel, T. VDAC and the bacterial
porin PorB of Neisseria gonorrhoeae share mitochondrial import pathways. EMBO J. 2002, 21,
1916–1929.
65. Massari, P.; Ho, Y.; Wetzler, L.M. Neisseria meningitidis porin PorB interacts with mitochondria
and protects cells from apoptosis. Proc. Natl. Acad. Sci. USA 2000, 97, 9070–9075.
66. Massari, P.; King, C.A.; Ho, A.Y.; Wetzler, L.M. Neisserial PorB is translocated to the
mitochondria of HeLa cells infected with Neisseria meningitidis and protects cells from apoptosis.
Cell. Microbiol. 2003, 5, 99–109.
67. Choi, C.H.; Lee, E.Y.; Lee, Y.C.; Park, T.I.; Kim, H.J.; Hyun, S.H.; Kim, S.A.; Lee, S.K.; Lee, J.C.
Outer membrane protein 38 of Acinetobacter baumannii localizes to the mitochondria and induces
apoptosis of epithelial cells. Cell. Microbiol. 2005, 7, 1127–1138.
68. Genestier, A.L.; Michallet, M.C.; Prevost, G.; Bellot, G.; Chalabreysse, L.; Peyrol, S.; Thivolet, F.;
Etienne, J.; Lina, G.; Vallette, F.M.; et al. Staphylococcus aureus Panton-Valentine leukocidin
directly targets mitochondria and induces Bax-independent apoptosis of human neutrophils. J. Clin.
Invest. 2005, 115, 3117–3127.
Int. J. Mol. Sci. 2012, 13 8050
69. Walther, D.M.; Bos, M.P.; Rapaport, D.; Tommassen, J. The mitochondrial porin, VDAC, has
retained the ability to be assembled in the bacterial outer membrane. Mol. Biol. Evol. 2010, 27,
887–895.
70. Danial, N.N.; Korsmeyer, S.J. Cell death: Critical control points. Cell 2004, 116, 205–219.
71. Rudel, T.; Kepp, O.; Kozjak-Pavlovic, V. Interactions between bacterial pathogens and
mitochondrial cell death pathways. Nat. Rev. Microbiol. 2010, 8, 693–705.
72. Jiang, J.H.; Tong, J.; Gabriel, K., Hijacking mitochondria: Bacterial toxins that modulate
mitochondrial function. IUBMB Life 2012, 64, 397-401.
73. Tanabe, M.; Nimigean, C.M.; Iverson, T.M. Structural basis for solute transport, nucleotide
regulation, and immunological recognition of Neisseria meningitidis PorB. Proc. Natl. Acad. Sci.
USA 2010, 107, 6811–6816.
74. Rudel, T.; Schmid, A.; Benz, R.; Kolb, H.A.; Lang, F.; Meyer, T.F. Modulation of Neisseria porin
(PorB) by cytosolic ATP/GTP of target cells: Parallels between pathogen accommodation and
mitochondrial endosymbiosis. Cell 1996, 85, 391–402.
75. Park, J.S.; Lee, W.C.; Yeo, K.J.; Ryu, K.S.; Kumarasiri, M.; Hesek, D.; Lee, M.; Mobashery, S.;
Song, J.H.; Kim, S.I.; et al. Mechanism of anchoring of OmpA protein to the cell wall
peptidoglycan of the gram-negative bacterial outer membrane. FASEB J. 2012, 26, 219–228.
76. Kaneko, J.; Kimura, T.; Narita, S.; Tomita, T.; Kamio, Y. Complete nucleotide sequence and
molecular characterization of the temperate staphylococcal bacteriophage phiPVL carrying
Panton-Valentine leukocidin genes. Gene 1998, 215, 57–67.
77. Yamashita, K.; Kawai, Y.; Tanaka, Y.; Hirano, N.; Kaneko, J.; Tomita, N.; Ohta, M.; Kamio, Y.;
Yao, M.; Tanaka, I. Crystal structure of the octameric pore of staphylococcal gamma-hemolysin
reveals the β-barrel pore formation mechanism by two components. Proc. Natl. Acad. Sci. USA
2011, 108, 17314–17319.
78. Roblin, P.; Guillet, V.; Joubert, O.; Keller, D.; Erard, M.; Maveyraud, L.; Prevost, G.; Mourey, L. A
covalent S-F heterodimer of leucotoxin reveals molecular plasticity of β-barrel pore-forming
toxins. Proteins 2008, 71, 485–496.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).
... The structures of BAM and SAM share little outside of the conserved core components BamA and Sam50 (Gentle et al., 2005;Jiang et al., 2012;Paschen et al., 2003;Ulrich & Rapaport, 2015). ...
... Yet, even with these differences, a commonality in BAM and SAM is observed, as some bacterial OMPs are able to utilize the mitochondrial SAM/TOM architecture for insertion into the OM and vice versa (Jiang et al., 2012). A structural alignment of monomeric and SAM, as well as OEP80, are all thought to share a common conserved mechanism for the biogenesis of βOMPs into the membrane. ...
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Gram‐negative bacteria, mitochondria, and chloroplasts all possess an outer membrane populated with a host of β‐barrel outer‐membrane proteins (bOMPs). These bOMPs play crucial roles in maintaining viability of their hosts and therefore, it is essential to understand the biogenesis of this class of membrane proteins. In recent years, significant structural and functional advancements have been made toward elucidating this process, which is mediated by the b‐barrel assembly machinery (BAM) in Gram‐negative bacteria, and by the sorting and assembly machinery (SAM) in mitochondria. Structures of both BAM and SAM have now been reported, allowing a comparison and dissection of the two machineries, with other studies reporting on functional aspects of each. Together, these new insights provide compelling support for the proposed budding mechanism, where each nascent bOMP forms a hybrid‐barrel intermediate with BAM/SAM in route to its biogenesis into the membrane. Here, we will review these recent studies and highlight their contributions towards understanding bOMP biogenesis in Gram‐negative bacteria and in mitochondria. We will also weigh the evidence supporting each of the two leading mechanistic models for how BAM/SAM function, and offer an outlook on future studies within the field.
... BamA is thought to be the workhorse of the complex, however, it is still not known exactly what role the other components play 17,18 . Orthologs of BamA can be found in eukaryotic organelles such as mitochondria (Sam50) and chloroplasts (Toc75/Oep80) where they are believed to perform a related function 2,5,15,[19][20][21][22][23][24][25] . ...
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In Gram-negative bacteria, the biogenesis of β-barrel outer membrane proteins is mediated by the β-barrel assembly machinery (BAM). The mechanism employed by BAM is complex and so far- incompletely understood. Here, we report the structures of BAM in nanodiscs, prepared using polar lipids and native membranes, where we observe an outward-open state. Mutations in the barrel domain of BamA reveal that plasticity in BAM is essential, particularly along the lateral seam of the barrel domain, which is further supported by molecular dynamics simulations that show conformational dynamics in BAM are modulated by the accessory proteins. We also report the structure of BAM in complex with EspP, which reveals an early folding intermediate where EspP threads from the underside of BAM and incorporates into the barrel domain of BamA, supporting a hybrid-barrel budding mechanism in which the substrate is folded into the membrane sequentially rather than as a single unit. The β-barrel assembly machinery (BAM) assists the folding and membrane insertion of bacterial outer membrane proteins. Here, the authors report structural characterization of BAM in lipid environment and in complex with the client protein EspP integrated into the barrel of BamA, providing insight into BAM mechanism of function.
... Interestingly, the formation and organization of OMPs in the bacterial membrane occurs mainly through the action of the Bam protein complex (b-Barrel Assembly Machinery), which is essential for bacterial survival [13]. Among these proteins, BamA represents a potential target, primarily due to the fact that it is anchored to the cell membrane, with a small extracellular portion that can generate immunogenic epitopes [14]. In addition, in silico and experimental analyses demonstrated that A. baumannii BamA is a good vaccine candidate [15,16]. ...
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Acinetobacter baumannii is an important nosocomial pathogen. BamA is a protein that belongs to a complex responsible for organizing the proteins on the bacterial outer membrane. In this work, we aimed to evaluate murine immune responses to BamA recombinant protein (rAbBamA) from A. baumannii in an animal model of infection, and to assess cross-reactivity of this target for the development of anti-A. baumannii vaccines or diagnostics. Immunization of mice with rAbBamA elicited high antibody titers and antibody recognition of native A. baumannii BamA. Immunofluorescence also detected binding to the bacterial surface. After challenge, immunized mice demonstrated a 40% survival increase and better bacterial clearance in kidneys. Immunoblot of anti-rAbBamA against other medically relevant bacteria showed binding to proteins of approximately 35 kDa in Klebsiella pneumoniae and Escherichia coli lysates, primarily identified as OmpA and OmpC, respectively. Altogether, our data show that anti-rAbBamA antibodies provide a protective response against A. baumannii infection in mice. However, the response elicited by immunization with rAbBamA is not completely specific to A. baumannii. Although a broad-spectrum vaccine that protects against various pathogens is an appealing strategy, antibody reactivity against the human microbiota is undesired. In fact, immunization with rAbBamA produced noticeable effects on the gut microbiota. However, the changes elicited were small and non-specific, given that no significant changes in the abundance of Proteobacteria were observed. Overall, rAbBamA is a promising target, but specificity must be considered in the development of immunological tools against A. baumannii.
... mitochondria, and chloroplast of eukaryotes, where their recognition, folding, and insertion are mediated by the BAM complex, SAM complex, and TOB complex, respectively. 1,4 Phylogenetic analysis indicates that these β-OMP assembly complexes contain a conserved Omp85 family protein, 2,5-8 suggesting that β-OMP biogenesis may share a similar mechanism in both Gram-negative bacteria and eukaryotes. ...
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β-barrel outer membrane proteins (β-OMPs) play critical roles in nutrition acquisition, protein import/export, and other fundamental biological processes. The assembly of β-OMPs in Gram-negative bacteria is mediated by the β-barrel assembly machinery (BAM) complex, yet its precise mechanism remains elusive. Here, we report two structures of the BAM complex in detergents and in nanodisks, and two crystal structures of the BAM complex with bound substrates. Structural analysis indicates that the membrane compositions surrounding the BAM complex could modulate its overall conformations, indicating low energy barriers between different conformational states and a highly dynamic nature of the BAM complex. Importantly, structures of the BAM complex with bound substrates and the related functional analysis show that the first β-strand of the BamA β-barrel (β1BamA ) in the BAM complex is associated with the last but not the first β-strand of a β-OMP substrate via antiparallel β-strand interactions. These observations are consistent with the β-signal hypothesis during β-OMP biogenesis, and suggest that the β1BamA strand in the BAM complex may interact with the last β-strand of an incoming β-OMP substrate upon their release from the chaperone-bound state.
... Samm50 is a component of the sorting and assembly machinery complex (SAM). It is in the outer membranes of mitochondria and has an essential role in the maintenance of mitochondrial cristae structure [34,35]. Samm50 targets contaminants and bacterial effector proteins that affect host mitochondria. ...
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The packaging of molecular constituents inside extracellular vesicles (EVs) allows them to participate in intercellular communication and the transfer of biological molecules, however the role of EVs during bacterial infection is poorly understood. The goal of this study was to examine the effects of Pseudomonas aeruginosa (P. aeruginosa) infection on the biogenesis and composition of EVs derived from the mouse microglia cell line, BV-2. BV-2 cells were cultured in exosome-free media and infected with 0, 1.3 × 104, or 2.6 × 104 colony forming units per milliliter P. aeruginosa for 72 h. The results indicated that compared with the control group, BV-2 cell viability significantly decreased after P. aeruginosa infection and BV-2-derived EVs concentration decreased significantly in the P. aeruginosa-infected group. P. aeruginosa infection significantly decreased chemokine ligand 4 messenger RNA in BV-2-derived infected EVs, compared with the control group (p ≤ 0.05). This study also revealed that heat shock protein 70 (p ≤ 0.05) and heat shock protein 90β (p ≤ 0.001) levels of expression within EVs increased after P. aeruginosa infection. EV treatment with EVs derived from P. aeruginosa infection reduced cell viability of BV-2 cells. P. aeruginosa infection alters the expression of specific proteins and mRNA in EVs. Our study suggests that P. aeruginosa infection modulates EV biogenesis and composition, which may influence bacterial pathogenesis and infection.
... Likely because of their bacterial origins, mitochondria and chloroplasts also possess two membranes with β-barrel proteins in their OMs (4). Outer-membrane proteins (OMPs) regulate traffic into and out of Gram-negative bacteria including water, ions, nutrients, and virulence factors in pathogenic bacteria; thus, they are critically important for bacterial survival (5)(6)(7)(8). Their surface exposure also makes them attractive antibiotic and vaccine targets, negating the need to breach one or both membranes. OMPs are synthesized in the cytoplasm, transported across the IM by the Sec translocon, and finally cross the periplasm with assistance from several chaperones (9)(10)(11)(12), before being assembled and inserted into the OM by the β-barrel assembly machinery (BAM) (13)(14)(15)(16)(17)(18)(19)(20). ...
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In Gram-negative bacteria, the outer membrane contains primarily β-barrel transmembrane proteins and lipoproteins. The insertion and assembly of β-barrel outer-membrane proteins (OMPs) is mediated by the β-barrel assembly machinery (BAM) complex, the core component of which is the 16-stranded transmembrane β-barrel BamA. Recent studies have indicated a possible role played by the seam between the first and last β-barrel strands of BamA in the OMP insertion process through lateral gating and a destabilized membrane region. In this study, we have determined the stability and dynamics of the lateral gate through over 12.5 μs of equilibrium simulations and 4 μs of free-energy calculations. From the equilibrium simulations, we have identified a persistent kink in the C-terminal strand and observed spontaneous lateral-gate separation in a mimic of the native bacterial outer membrane. Free-energy calculations of lateral gate opening revealed a significantly lower barrier to opening in the C-terminal kinked conformation; mutagenesis experiments confirm the relevance of C-terminal kinking to BamA structure and function.
... An interesting consequence of the evolution of the bacterial BAM complex into the SAM complex in eukaryotes is that pathogens (Gram-negative bacteria) may use the eukaryotic host machinery to fold and assemble their proteins in the mitochondrial outer membrane [101]. For example, bacterial toxins that target mitochondria are folded by the SAM complex. ...
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The biogenesis of transmembrane β-barrels (outer membrane proteins, or OMPs) is an elaborate multistep orchestration of the nascent polypeptide with translocases, barrel assembly machinery, and helper chaperone proteins. Several theories exist that describe the mechanism of chaperone-assisted OMP assembly in vivo and unassisted (spontaneous) folding in vitro. Structurally, OMPs of bacterial origin possess even-numbered strands, while mitochondrial β-barrels are even- and odd-stranded. Several underlying similarities between prokaryotic and eukaryotic β-barrels and their folding machinery are known; yet, the link in their evolutionary origin is unclear. While OMPs exhibit diversity in sequence and function, they share similar biophysical attributes and structure. Similarly, it is important to understand the intricate OMP assembly mechanism, particularly in eukaryotic β-barrels that have evolved to perform more complex functions. Here, we deliberate known facets of β-barrel evolution, folding, and stability, and attempt to highlight outstanding questions in β-barrel biogenesis and proteostasis.
... Notably, the signal targeting bacterial beta-barrel proteins to the outer membrane of Gram-negative bacteria are functional in eukaryotic mitochondria, indicating a conservation of the targeting pathway (Walther et al. 2009a(Walther et al. , 2009b. Considering the prokaryotic origin of the mitochondrion, we see that bacteria can probably exploit this evolutionary relationship during infection to direct virulence factors to mitochondria (Jiang et al. 2012b;Lucattini et al. 2004). For instance, the recognition and import of the proapoptotic protein PorB, an ATP-binding beta-barrel porin encoded by Neisseria meningitidis, may be attributable to structural similarities between it and the endogenous mitochondrial porin called the voltage-dependent anion-selective channel (VDAC; Jiang et al. 2011;Muller et al. 2000Muller et al. , 2002. ...
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Manipulation of host cell function by bacterial pathogens is paramount for successful invasion and creation of a niche conducive to bacterial replication. Mitochondria play a role in many important cellular processes including energy production, cellular calcium homeostasis, lipid metabolism, haeme biosynthesis, immune signalling and apoptosis. The sophisticated integration of host cell processes by the mitochondrion have seen it emerge as a key target during bacterial infection of human host cells. This review highlights the targeting and interaction of this dynamic organelle by intravacuolar bacterial pathogens and the way that the modulation of mitochondrial function might contribute to pathogenesis.
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Background Outer membrane vesicles (OMVs) of Acinetobacter baumannii are cytotoxic and elicit a potent innate immune response. OMVs were first identified in A. baumannii DU202, an extensively drug-resistant clinical strain. Herein, we investigated protein components of A. baumannii DU202 OMVs following antibiotic treatment by proteogenomic analysis. Methods Purified OMVs from A. baumannii DU202 grown in different antibiotic culture conditions were screened for pathogenic and immunogenic effects, and subjected to quantitative proteomic analysis by one-dimensional electrophoresis and liquid chromatography combined with tandem mass spectrometry (1DE-LC-MS/MS). Protein components modulated by imipenem were identified and discussed. ResultsOMV secretion was increased > twofold following imipenem treatment, and cytotoxicity toward A549 human lung carcinoma cells was elevated. A total of 277 proteins were identified as components of OMVs by imipenem treatment, among which β-lactamase OXA-23, various proteases, outer membrane proteins, β-barrel assembly machine proteins, peptidyl-prolyl cis–trans isomerases and inherent prophage head subunit proteins were significantly upregulated. Conclusion In vitro stress such as antibiotic treatment can modulate proteome components in A. baumannii OMVs and thereby influence pathogenicity.
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In Gram-negative bacteria, the biogenesis of β-barrel outer membrane proteins (OMPs) is mediated by the β-barrel assembly machinery (BAM) complex. During the past decade, structural and functional studies have collectively contributed to advancing our understanding of the structure and function of the BAM complex; however, the exact mechanism that is involved remains elusive. In this Progress article, we discuss recent structural studies that have revealed that the accessory proteins may regulate essential unprecedented conformational changes in the core component BamA during function. We also detail the mechanistic insights that have been gained from structural data, mutagenesis studies and molecular dynamics simulations, and explore two emerging models for the BAM-mediated biogenesis of OMPs in bacteria.
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Voltage-dependent anion-selective channel (VDAC) is a β-barrel protein in the outer mitochondrial membrane that is necessary for metabolite exchange with the cytosol and is proposed to be involved in certain forms of apoptosis. We studied the biogenesis of VDAC in human mitochondria by depleting the components of the mitochondrial import machinery by using RNA interference. Here, we show the importance of the translocase of the outer mitochondrial membrane (TOM) complex in the import of the VDAC precursor. The deletion of Sam50, the central component of the sorting and assembly machinery (SAM), led to both a strong defect in the assembly of VDAC and a reduction in the steady-state level of VDAC. Metaxin 2-depleted mitochondria had reduced levels of metaxin 1 and were deficient in import and assembly of VDAC and Tom40, but not of three matrix-targeted precursors. We also observed a reduction in the levels of metaxin 1 and metaxin 2 in Sam50-depleted mitochondria, implying a connection between these three proteins, although Sam50 and metaxins seemed to be in different complexes. We conclude that the pathway of VDAC biogenesis in human mitochondria involves the TOM complex, Sam50 and metaxins, and that it is evolutionarily conserved.
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The preprotein translocase of the outer mitochondrial membrane (TOM complex) contains one essential subunit, the channel Tom40. The assembly pathway of the precursor of Tom40 involves the TOM complex and the sorting and assembly machinery (SAM complex) with the non-essential subunit Mas37. We have identified Sam50, the second essential protein of the mitochondrial outer membrane. Sam50 contains a beta-barrel domain conserved from bacteria to man and is a subunit of the SAM complex. Yeast mutants of Sam50 are defective in the assembly pathways of Tom40 and the abundant outer membrane protein porin, while the import of matrix proteins is not affected. Thus the protein sorting and assembly machinery of the mitochondrial outer membrane involves an essential, conserved protein.
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