Candidacy Proposal

Program Director/Principal Investigator (Schonborn, Wesley, Adam):

SPECIFIC AIMS:

Acinetobacter baumannii is an emerging nosocomial pathogen. It is inherently resistant to some β-
lactams. Many strains are also resistant to aminoglycosides, fluoroquinolones, tetracyclines and
polymyxins. It is highly competent and resistant to other classes of antibiotics (Peleg, Seifert et al.
2008). There are only a few effective antibiotics against A. baumannii such as Tigecycline, colistin
and sulbactam, however there are sporadic instances of resistance to even these antibiotics and they
may themselves toxic to the patient. Efflux are an emerging area of discovery on the multidrug
resistance front. There are only three characterized efflux pumps in A. baumannii: adeABC, adeIJK
and abeM (Schneiders, Findlay et al. 2008) However, a genetic screen has shown more than 30
putative efflux pumps (Fournier, Vallenet et al. 2006) from several families, potentially encoding
resistance to dozens of antibiotics. Overcoming antibiotic resistance mediated by efflux pumps
requires a two-pronged approach consisting of (1) identifying all efflux pumps present in a bacterium
and (2) blocking the function of these efflux pumps. Therefore, I propose to elucidate the function
of two promising putative MATE family efflux pumps with known efflux pump inhibitors and
determine whether a number of potential MATE inhibitors will interrupt their function.

1) Investigate the function of newly found genes that code for probable MATE family efflux
pumps.
• Determine which antibiotics are substrates.
• Investigate the effect of removing or adding these pumps using an E. coli model system.
• Investigate the effect on virulence factors such as biofilm formation with deletion of the
efflux genes.

2) Determine the ability of several compounds to act as MATE inhibitors and reduce the
effectiveness of these efflux pumps.
• Determine the synergistic effects of these compounds on normally ineffective (due to being
MATE substrates) antibiotics.
• Identify up and downregulation of known and newly identified efflux pumps in response to
compound administration.
• Determine binding affinity of compounds to efflux pumps.

Since antibiotic resistance of A. baumannii has accelerated in the past decade this sort of research is
desperately needed (Munoz-Price and Weinstein 2008).

PHS 398/2590 (Rev. 11/07) Page Continuation Format Page


TITLE: The characterization of putative efflux pumps in Acinetobacter baumannii and a
screen of compounds to inhibit these pumps.

BACKGROUND AND SIGNIFICANCE:

Bacteria have been resisting antibiotics since before humans discovered penicillin. Antibiotic
resistance is a worldwide problem that has been occurring since the discovery of antibiotics in the
1930s and is being seen more and more in gram-negative bacteria (Poole 2004). The overuse of
antibiotics in the hospital and even farm setting (Levy, FitzGerald et al. 1976) has created a perfect
storm of selective pressure for bacteria to become resistant. Multiple drug resistance (MDR) is
becoming problematic since its original discovery in enteric bacteria in the 1950s and 1960s (Levy
and Marshall 2004). Now it is moving into other bacteria including non-pathogenic communal
species. Multiple drug resistance (MDR) is the most troubling aspect as it may require up to seven
antibiotics to be used concomitantly which risks the health of the host (Iseman 1993). This could also
breed resistance to these last line antibiotics, many of which are toxic. The variety of organisms,
breadth of geographic areas and multidrug resistance characteristics have become increasingly
worrying (Levy 1998).

One of the most infamous of the MDR bacteria is methicillin resistant Staphylococcus aureus
(MRSA). In the hospital setting 40-60% of the time it is MDR (Weinstein 2001). Although MRSA gets
a lot of attention, there are other bacteria that also cause nosocomial infections. Some of the most
severe is Acinetobacter baumannii and Pseudomonas aeruginosa which are usually resistant to all
but one antibiotic in the hospital setting. A. baumannii is particularly worrisome due to a 32-64%
mortality rate (book page 62). Acinetobacter baumannii is inherently resistant to β-lactam antibiotics.
A. baumannii is naturally competent (Levy and Marshall 2004; Vallenet, Nordmann et al. 2008)
making them particularly problematic.

Acinetobacter baumannii is a gram-negative coccobacillus. It is strictly aerobic, non-fermenting,
nonfastidious, nonmotile, catalase-positive, oxidase-negative bacteria with a DNA GC content of 39%
to 47% (Peleg, Adams et al. 2007). The members of the Genus Acinetobacter have changed over the
past several decades and species names have also changed, complicating Acinetobacter research. It
is a gram-negative aerobic coccobacillus that was first identified in 1911 as Micrococcus calco-
aceticus (Munoz-Price and Weinstein 2008). It was named Acinetobacter in the 1950s.
Acinetobacter baumannii is difficult to differentiate from other Acinetobacter species using common
biochemical tests. The only sure method of identifying A. baumannii is by DNA-DNA hybridization.
A. baumannii is responsible for pneumonia, bloodstream infections, urinary tract infections and even
nosocomial meningitis (Peleg, Seifert et al. 2008). The Acinetobacter genus now consists of 32
species, only 17 of which have been named. There are two other closely related types known as
genomic species 3 and genomic species 13TU which are mostly indistinguishable from each other
and so are put into a group known by the term “Acb-complex” (Dijkshoorn 2008). Three
Acinetobacters were recently sequenced and compared: A. baumannii AYE, A. baumannii SDF and
A. baylyi ADP1 (Vallenet, Nordmann et al. 2008). AYE and SDF are both human pathogens but SDF
is susceptible to antibiotics. A. baylyi ADP1 was used as a reference and is a nonpathogenic soil-
dwelling Acinetobacter species. AYE has an antibiotic resistance island of about 86kb and 52
predicted genes associated with resistance within and without the island (Fournier, Vallenet et al.
2006; Vallenet, Nordmann et al. 2008). There are more than 30 putative efflux pumps in A.
baumannii AYE (Fournier, Vallenet et al. 2006).



Bacterial efflux pumps are a normal part of bacteria that transport molecules out of the periplasmic
space of gram-negative bacteria. They may be encoded chromosomally or on plasmids and also
may be induced by the molecule being pumped out. There are five families of efflux pumps:
multidrug and toxic compound extrusion (MATE), major facilitator superfamily (MFS), staphylococcal
multiresistance (SMR), resistance nodulation division (RND) and the ATP-binding cassette (ABC)
superfamily. MFS efflux pumps have been examined the most; NorA in S. aureus and PmrA in
Streptococcus pneumoniae (Piddock 2006). They have 12 transmembrane regions and operate via
the proton motive force (PMF) (Yoshida, Bogaki et al. 1990; Gibbons, Oluwatuyi et al. 2003). SMR
blah blah blah. ABC efflux pumps are known to transport molecules but their role in antibiotic
resistance has not been found (Piddock 2006). MATE pumps have been found in a number of
important bacteria including S. aureus and Vibrio cholerae (Piddock 2006).

MATE proteins are responsible for multidrug resistance in Acinetobacter baumannii as well as other
prokaryotes, eukaryotes and the Achaea (Omote, Hiasa et
al. 2006). They function on a electrochemical gradient of
H+ or Na+ (Omote, Hiasa et al. 2006). Originally they
were grouped with the MFS family of transporters because
of their similarity in having 12 transmembrane helices
(Morita, Kodama et al. 1998). However, it was then found
that they did not show any sequence identity to known
MFS transporters and were therefore given a new name,
multidrug and toxic compound extrusion (Brown, Paulsen
et al. 1999). Analysis of sequences showed that all MATE
proteins are likely descended from a common ancestor
that underwent gene duplication (Otsuka, Matsumoto et al.
2005). Human MATE proteins exist and are responsible
for pumping out toxic compounds as well. This
demonstrates the need for research on these important
pumps because they may pump out drugs needed to kill
cancerous cells. Until now, only one MATE protein, abeM,
has been characterized in A. baumannii (Su, Chen et al. 2005). Characterizing two more will aid in
finding antibiotics or efflux pump inhibitors that can help contain A. baumannii infection.

Finding efflux pumps and characterizing them is the first step in using them against bacteria. The
next step is to inhibit them so that the bacteria can not extrude what they need to whether it is a
natural part of life or an antibiotic. Recently, several novel antibiotics have been found that may act
by inhibiting efflux pumps. The molecular mechanisms of how these antibiotic (and possible efflux
inhibiting) molecules is not known. Studying them will lead directly to therapy for disease caused by
A. baumannii. Also, the more that is known about them, the more drugs can be rationally designed to
target them or avoid them (Poole 2004). One of these, (-)-epigallocatechin-3-gallate (EGCg) from
green tea, has been found to have antibacterial activity against A. baumannii in vitro (Osterburg,
Gardner et al. 2009). However, the molecular mechanisms behind this activity have not yet been
elucidated. EGCg has been shown to allow β-lactam sensitivity in Pseudomonas aeruginosa and
Staphylococcus aureus; this synergism is an indication that it works by blocking efflux pumps. Others
have found that it also interrupts peptidoglycan synthesis (Zhao, Hu et al. 2001) in S. aureus however
A. baumannii is gram-negative and may not be as effected by peptidoglycan issues. Recently, a
number of other phytochemicals (Piperine, Quinine and Harmaline) have been found to be efflux
pump inhibitors (Mohtar, Johari et al. 2009). Piperine was found to inhibit efflux in S. aureus.



SPECIFIC AIM 1. Investigate the function of newly found genes that code for probable antibiotic
resistance proteins.

Rationale.

Studies have shown that acquiring a new efflux pump is the first step toward becoming resistant to
additional antibiotics (Oethinger, Kern et al. 2000). So finding a way to disable the efflux pumps will
help in two ways: first, the pump itself will not be able to remove antibiotic and second, the bacterium
will not have as much time available to either upregulate other pumps or to divide and eventually
become resistant to other antibiotics. In fact, reserpine has been found to block efflux pumps in S.
aureus and prevent it from becoming resistant to norfloxacin (Markham and Neyfakh 1996). Other
antibiotics and bacterial species have also been tested in this manner (Markham 1999; Markham,
Westhaus et al. 1999; Lomovskaya, Warren et al. 2001).

A genetic screen has found many possible efflux pumps. These are identified due to their
resemblance to known efflux pump motifs. Two were found that fit into the MATE family of efflux
proteins. One of these, 36_127, is also found in Acinetobacter spp. ADP1 which has been fully
sequenced. Another, 36_503, is not found in ADP1 but may be from Bordetella bronchiseptica
(Fournier, Vallenet et al. 2006). Studying both of these will show the function of a pump in highly drug
resistant and normal A. baumannii. Since A. baumannii is highly competent it is likely for another
strain to also be able to pick up 36_503 from either B. bronchiseptica or A. baumannii harboring the
gene. Moving the gene from the native bacterium to E. coli missing its efflux pumps (AcrAB and
YdhE) will allow each pump to be studied independently of any other pumps. The antibiotics chosen
represent members of the major classes of antibiotics as well as some other molecules, such as
ethidium bromide, which are know to be substrates for efflux pumps.

There are three known and characterized RND efflux pumps in A. baumannii. Many of these are also
found in less virulent strains such as SDF and non-pathogenic (ADP1) so they deserve an intense
look so as to find drugs that may inhibit them. RND efflux pumps consist of three parts: the inner
membrane transporter (efflux) protein, an outer membrane protein and a periplasmic accessory
protein mediating between the inner and outer membrane proteins. This complicates discovery of
function since the three that are together are not necessary in a single operon and yet need to be
studied together. There is also adeXYZ but adeY has not been successfully disrupted likely because
it has essential cellular functions (Chu, Chau et al. 2006). As such, adeXYZ will not be studied.

Hypothesis.

Two putative MATE efflux pump genes in Acinetobacter baumannii are responsible for efflux of a
variety of antibiotics.

Overview of Experimental Approach.

1. The first experimental step will be to design primers for the two genes to be studied. The
sequence of these genes is available on the EMBL website under CT025821 and CT025818
(http://www.ebi.ac.uk/). Once designed the gene will be cloned out and ligated into pBR322.
Amplification will add a BamHI to either end in order to clone into pBR322. Colonies will be
spread on plates containing tetracycline in order to select for positive clones. The plasmids can
then be extracted using a commercial mini-prep kit (Qiagen) and Escherichia coli KAM32


transformed with them. Each gene will be separately placed on a plasmid (pWS100?) and
electroporated into E. coli with an empty pBR322 used as a control (Su, Chen et al. 2005).
2. The susceptibility to a variety of antibiotics will be compared between the efflux pump carrying
E. coli and the control. Antibiotics representing all of the major antibiotic classes will be used
(Table 1) as well as other known efflux pump substrates. Susceptibility will be assessed using
a 2-fold dilution method in Mueller-Hinton broth (Begum, Rahman et al. 2005; Damier-Piolle,
Magnet et al. 2008) in a 96-well plate to allow many replications and antibiotics. The minimum
inhibitory concentration (MIC) is defined as the lowest concentration of antibiotic used that
prevented bacterial growth.
3. Accumulation or efflux of ethidium bromide will then be measured by fluorescence intensity
(Xu, Su et al. 2003).
4. Another characteristic of MATE pumps is that they are Na+-antiporters, therefore, efflux of a
known substrate (from previous experiments) will be measured with varying concentrations of
Na+ or K+ (Long, Rouquette-Loughlin et al. 2008).
5. Targeted deletion of the MATE genes. This will show if they are a necessary part of natural
biological function for A. baumannii.

Expected Results.

It is expected that the recombinant pumps from A. baumannii will confer resistance to antibiotics in E.
coli. The MIC will increase if the pump is pumping out a particular antibiotic. The substrate for either
of the pumps in the fluorescent assays will show lower fluorescence intensity within cells over time
due to the pumping out of fluorescent molecules.

1.
2.
3.
4.
5.
6.
7.

Potential Pitfalls.

It is possible that the E. coli cells chosen will not transform. There are other strains available that are
AcrAB deficient: AG100AX, AG100A and KAM3 (Mine, Morita et al. 1999; Xu, Su et al. 2003; Long,
Rouquette-Loughlin et al. 2008). If the pBR322 plasmid suffers from problems during cloning pUC19
can also be used and should give identical results in E. coli (Begum, Rahman et al. 2005).
Susceptibility can also be detected using the disk diffusion method on Mueller-Hinton agar plates or
regular sized test tubes if the 96-well plate method does not work. If ethidium bromide turns out not
to be a substrate for either pump, another fluorescent molecule can be used for the fluorescence
assays: Hoechst 33342, rhodamine 6G, proflavine as well as the antibiotics norfloxacin and
ciprofloxacin (Long, Rouquette-Loughlin et al. 2008). It is also possible that these are not MATE
efflux pumps; they may be pseudogenes.



SPECIFIC AIM 2. Investigate the ability of several putative efflux pump inhibitors to interfere with the
function of AbeM and the two newly characterized MATE efflux pumps.

Rationale

These compounds are known to be antibiotic but their mechanism of action is not understood.
Synergy with another antibiotic and having a pump inhibitor ability in Pseudomonas aeruginosa gives
an indication that they will block efflux pumps in A. baumannii.

Hypothesis.

Several recently identified compounds will interfere with the function of the known MATE efflux
pump, AbeM as well as the two newly characterized MATE efflux pumps and restore susceptibility
to antibiotics.

Overview of Experimental Approach.

1. Find MIC of new compounds; or whether they even have a MIC.
2. .
3. Ethidium bromide accumulation assay
4. Find MIC of ineffective antibiotics in the presence of the new compounds (MIC synergy
testing).
5. FIC assay to determine dosage.
6. Protonophore assay to assure that the compounds are not merely interrupting the proton
motive force (make sure PMF is involved with MATE pumps).
7. Perform binding studies to determine how the compounds are disabling the efflux pumps.
8. Perform mutagenesis to discover which motifs/residues are important for binding of the
compounds.

Potential Pitfalls

Expected Results

Experimental Section:



References

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