IAI Accepts, published online ahead of print on 23 October 2006 Infect. Immun. doi:10.1128/IAI.01226-06 Copyright © 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
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AcpA: A Francisella Acid Phosphatase Affecting
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Intramacrophage Survival and Virulence
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Nrusingh P. Mohapatra, Ashwin Balagopal, Shilpa Soni, Larry S. Schlesinger, and John
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S. Gunn*
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The Center for Microbial Interface Biology; Department of Molecular Virology,
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Immunology, and Medical Genetics; and Department of Internal Medicine, Division of
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Infectious Diseases, The Ohio State University, Columbus, OH 43210 USA
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Running title: Francisella acid phosphatase AcpA
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*corresponding author
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The Ohio State University
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Tzagournis Medical Research Facility
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420 W. 12th Ave.
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Columbus, OH 43210-1214
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Phone: 614-292-6036
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Fax: 614-292-5495
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E-mail:
[email protected]
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Abstract
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AcpA of Francisella spp. is a respiratory burst-inhibiting acid phosphatase that also
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exhibits phospholipase C activity. To better understand the molecular basis of AcpA in
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virulence, a deletion of acpA was constructed in Francisella novicida. The phosphatase
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and lipase activities were reduced 10-fold and 8-fold, respectively, in the acpA mutant
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versus the wild type, and were found mostly associated with the outer membrane. The
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acpA mutant was more susceptible to intracellular killing than the wild type strain in the
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THP-1 human macrophage-like cell line. In addition, mice infected with the acpA mutant
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survived longer than the wild type strain and were less fit than the wild type strain in
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competition infection assays. Transmission electron microscopy showed that the acpA
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mutant was delayed in escape from macrophage phagosomes, as more than 75% of acpA
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mutant bacteria could still be found inside phagosomes after 12 hrs of infection in THP-1
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cells and human monocyte-derived macrophages, whereas most of the wild type bacteria
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had escaped from the phagosome by 6 hrs post-infection. Thus, AcpA affects intracellular
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trafficking and the fate of Francisella within host macrophages.
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Introduction 1
Francisella tularensis is a gram negative, facultative intracellular pathogen that causes
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tularemia in humans and other mammals including rodents (38). The two primary human
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pathogens are F. tularensis subspecies tularensis (type A strain) and F. tularensis
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subspecies holarctica (type B strain). The type A strain is predominantly found in North
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America, highly infectious and causes a life threatening disease in humans, especially
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when inhaled (10). Type B strains are found primarily in Europe and are considered to be
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less virulent for mammals than the type A strain (10). An attenuated live vaccine strain
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(LVS) was derived from a type B strain (12) and it elicits a protective response in
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humans, monkeys, guinea pigs and mice against systemic challenge with virulent type A
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F. tularensis (9, 12, 13, 18, 35, 36). Francisella novicida (FnWT), a close relative of type
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A F. tularensis, is a much less frequent cause of infection in humans than either type A or
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type B (19). Nevertheless, F. novicida and F. tularensis subspecies tularensis and
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holarctica all cause a lethal systemic infection in mice when inoculated by most routes
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(19).
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The virulence mechanisms of this bacterium are not clear, although the products
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of several genes such as mglA and the pathogenicity island genes iglC, iglD and pdpA-D
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have been identified that help Francisella to survive inside macrophages (6, 15, 17).
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However, the exact functions of these genes are not known. MglA shares homology with
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the SspA of E. coli, which regulates the stationary phase gene transcription by interacting
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with RNA polymerase (40). MglA regulates several virulence factors within the
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pathogenicity island including iglC (25), which is important for the ability of Francisella
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spp. to escape from the phagosome. Several studies have shown that F. tularensis
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resides inside a membrane-bound phagosome during its initial growth in a macrophage
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and that it is released into the cytoplasm during a later phase of growth (2, 11, 16).
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Acid phosphatases are ubiquitous in nature and are present in almost all bacteria.
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These enzymes have been identified and characterized from many eukaryotes and
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prokaryotes and divided into subgroups according to their substrate specificity, molecular
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weight and sensitivity to known inhibitors (30). Acid phosphatases catalyze the
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hydrolysis of phospho-monoesters at an acidic pH. In several species they have been
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implicated as virulence factors and help the bacteria to survive inside phagocytes (4, 7,
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14, 24, 27, 28, 31), often by inhibiting the respiratory burst (4, 20, 24, 29, 31).
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The published genome sequence of F. tularensis Schu 4 revealed the presence of
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four acid phosphatases (acpA, FTT0221; acpB, FTT0156; acpC, FTT0620; and hap,
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FTT1662c [a pseudogene in Schu 4 but not LVS]) (21). AcpA (57 kDa) is a polyspecific
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periplasmic acid phosphatase that is highly expressed by F. tularensis (7, 27) and shows
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no significant global amino acid sequence similarity with any protein in the Protein Data
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Bank (8). This protein is also unusual in that it exhibits phospholipase C activity (27).
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Earlier studies reported that Francisella AcpA has respiratory burst inhibiting properties
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and broad substrate specificity (27). It has also been shown that a transposon insertion in
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the 3’ region of the acpA open reading frame did not result in an intramacrophage
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survival defect or a loss of virulence (7). In the present study, we constructed a deletion
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of the entire acpA gene in FnWT and analyzed its role in intracellular trafficking in
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macrophages and virulence in mice.
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Materials and Methods 1
Bacterial Strain, Plasmid Construction and Molecular Biology Techniques
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Bacterial strains, plasmids and primers used in this study are listed in Table-1. F.
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novicida U112 was routinely grown at 37°C in cysteine heart agar (CHA) (Hi-Media
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Laboratories, India) and modified Tryptic Soy Broth (TSB) (Difco Laboratories, Detroit,
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Mich.) containing 135ug/ml ferric pyrophosphate and 0.1% cysteine hydrochloride. CHA
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containing 5% defibrinated sheep blood and 135ug/ml ferric pyrophosphate was used for
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transformation studies (Hemostat Laboratories, Dixon, Calif.). When required, the growth
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medium for FnWT was supplemented with kanamycin (25 µg/ml) or tetracycline (12.5
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µg/ml). All manipulations with Francisella spp. were performed in a class II biological
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safety laboratory. E. coli DH5α was grown at 37°C aerobically in Luria-Bertani (LB)
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medium (Difco Laboratories, Detroit, Mich.) supplemented with kanamycin (15 µg/ml),
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tetracycline (12.5 µg/ml), or ampicillin (100 µg/ml) when required. All antibiotics and
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chemicals were purchased from Sigma-Aldrich (St. Louis, Mo.).
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Chromosomal DNA preparation, ligation, E. coli transformation, and Southern
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blotting were performed according to methods described by Sambrook et al (32). To
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construct the pAcpA-Kan plasmid, upstream and downstream regions of acpA were
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generated by PCR with primers JG996/JG997 and JG998/JG999 respectively from FnWT
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genomic DNA. The 772-bp upstream fragment was digested with SacI and KpnI, cloned
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into dephosphorylated SacI and KpnI digested pUC18, and renamed pAcpUp. Similarly
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871-bp downstream region of acpA was digested with PstI and SphI, cloned into
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dephosphorylated PstI and SphI digested pAcpUp plasmid, and renamed pAcpUpDn. A
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Kan cassette driven by the F. tularensis outer membrane protein (YP_169847) promoter
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was amplified from plasmid pDSK519 using primers JG868 /JG1048, digested with KpnI
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and PstI, ligated into KpnI/PstI digested and dephosphorylated pAcpUpDn vector, and
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renamed pAcpA-Kan. Plasmid pAcpA-Kan was transformed into FnWT by
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cryotransformation. In brief, 1 µg of plasmid was mixed with FnWT cells at an OD600 of
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0.5 in transformation buffer (10mM MOPS, 75mM CaCl2, 10mM RbCl2 and 15%
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glycerol, pH adjusted to 6.5 with 1N KOH), incubated on ice for 30 min. and flash frozen
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in liquid nitrogen for 5 min. Cells were thawed at room temperature and plated on CHA-
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Kan25 and incubated at 37° C. This resulted in the creation of strain JSG2660
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(∆acpA::kan). Growth curves were identical for strains JSG2660 and FnWT. For
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complementation in trans, plasmid pKK214 containing the groEL promoter of F.
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tularensis LVS was used (1). The acpA gene was generated by PCR using primers
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JG1016a/JG1017a and cloned into the EcoRI and PstI sites of pKK214groEL such that
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acpA was expressed from the groEL promoter. The resulting plasmid pAcpA, was
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introduced into JSG2660 by cryotransformation described as above creating strain
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JSG2661.
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For Southern blot analysis, PCR products were labeled with digoxigenin as
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instructed by the manufacturer (Roche, Indianpolis, USA). Probes were hybridized to
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EcoRI digested chromosomal DNA of FnWT and Fn∆acpA followed by anti-
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digoxigenin-AP-conjugate antibody treatment. Membranes were developed with
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NBT/BCIP solution.
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Cell fractionation
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Overnight stationary phase cultures of wild type (JSG1819) and mutant (JSG2660)
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bacteria in TSB-0.1% cysteine hydrochloride were centrifuged at 8000 x g for 20 min at
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4° C. The cell pellet was washed twice with PBS and sonicated at a constant output of
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60 W for a total of 300 s. Cell debris and unbroken cells were removed by centrifugation
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at 3000 x g for 15 min at 4° C. The supernatant was centrifuged at 25,000 x g for 24 h at
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18° C in a 2.1::1.4::0.7 M sucrose gradient to separate the outer and inner membrane
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fractions. The non-membrane containing fraction was used as the cytosolic fraction.
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These fractions were assayed for phosphatase activity as described by Aragon et al. (3)
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Western blot analysis was performed by standard protocols on protein fractions using
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anti-AcpA polyclonal sera (gift of Tom Reilly).
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Intramacrophage Survival Assays
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FnWT or Fn∆acpA strains were used to infect the following cell lines: J774.1 murine
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macrophages, NR8383 rat alveolar macrophages, and PMA-induced (10 ng/ml) THP-1
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human macrophages at an MOI of ~50:1. Wells were seeded with ~2 x105 macrophages
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and ~1.0x107 bacteria were added to each well. After 2 h incubation at 37° C, 5% CO2,
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gentamicin (50 µg/ml) was added to the medium to eliminate extracellular organisms.
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Wells were washed twice with PBS and incubated with their respective media
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supplemented with 10 µg/ml gentamicin. The macrophage cells were lysed with 0.1%
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SDS at 2h, 12h and 24 h post infection, the lysates were immediately serially diluted in
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PBS and plated on CHA plates for determination of viable counts. Experiments were
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performed in triplicate on a minimum of three separate occasions with similar results.
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Transmission Electron Microscopy
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Monolayers of monocyte-derived macrophages (MDMs) (23) or THP-1 cell lines were
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incubated with FnWT (JSG1819) or Fn∆acpA (JSG2660) at an MOI of 500:1 in a plastic
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four-well chamber slide. This high MOI was used to enhance the number of macrophages
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in the population that were infected, which in turn aided visualization. After 2, 6 and 12
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hrs of incubation at 370 C, 5% CO2, the wells were washed and fixed immediately with
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2.5% warmed glutaraldehyde for 5 min followed by a combination of 2.5%
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glutaraldehyde and 1% osmium tetroxide in 0.1M sodium cacodylate, pH 7.3 for 15 min
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at 4° C (11). The cells were then stained with 0.25% uranyl acetate in a 0.1M sodium
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acetate buffer at pH 6.3 for 45 minutes. Monolayers were washed with ice cold normal
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saline and the chambers were then removed. Slides were dehydrated through a graded
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series of ethanol, rinsed in HPMA (hydroxypropylmethylacrylate) and infiltrated with
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Polybed 812. They were embedded by up-ending resin-filled beam capsules over the
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cells and polymerized at 60° C for 24 h. Thin sections cut with a Leica EM UC6
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ultramicrotome were collected on Formvar coated copper grids, stained with uranyl
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acetate and lead citrate and viewed by transmission electron microscopy in a Philips
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CM12 at 60 kV. Multiple fields were examined for bacteria, and identified bacteria were
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determined to be intraphagosomal or intracytosolic. The criterion for intraphagosomal
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was the visualization of greater than 50% of the phagosomal membrane surrounding the
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bacterium.
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Mouse Survival Studies
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Groups of five female 4 to 6 week-old BALB/c mice (Harlan Sprague) were inoculated at
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a dose of ~100 CFU delivered in 100 µl PBS by the intraperitoneal route. Actual bacterial
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counts delivered were determined by plate count from each inoculum. Mice were
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monitored for four days post-infection. For competition assays, wild type (JSG1819) and
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mutant (JSG2660) bacteria were inoculated into BALB/c mice at a 1:1 ratio (100 CFU of
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each in 100 µl total) and at four days post inoculation, organs were harvested, macerated,
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and plated onto appropriate solid media to select for each of the competing strains. The
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competitive index was calculated as the number of mutant CFU/FnWT CFU recovered.
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Results
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Construction of a Fn∆acpA mutant
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A modified cryotransformation technique was developed that increased the efficiency of
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transformation 100-1000 fold in comparison to the standard Francisella MgCl2-KCl
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cryotransformation or electroporation. This technique was used to introduce pAcpA-Kan,
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a non-replicating suicide vector carrying a DNA fragment with a kanamycin cassette in
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the place of a complete deletion of acpA, into FnWT. After 5-days growth on CHA-Kan25
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plates, several colonies were screened by PCR and Southern blot resulting in the
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identification of more than 85% of the clones with the correct deletion and loss of the
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vector sequences (Figure 1A).
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Comparative measurements of acid phosphatase and lipase activity
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FnWT and Fn∆acpA cells were disrupted and separated by sucrose gradient to recover
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inner and outer membrane fractions. Western blot analysis was performed which showed
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the presence of AcpA in whole cell lysates and the outer membrane fraction of FnWT
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(Figure 1B). AcpA was absent in all fractions analyzed from the Fn∆acpA strain. A
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single band of similar molecular weight was detected in a Schu 4 whole cell lysate,
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demonstrating its expression in Type A virulent Francisella. Acid phosphatase activities
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of the Fn cell-free extracts and inner and outer membrane fractions were measured.
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Using 6,8-difluoro-4-methylumbelliferyl phosphate as the substrate, we found no
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difference in phosphatase activities of cell-free extracts/cytosolic fractions or inner
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membrane fractions between the mutant and wild-type strains, but a 10-fold decrease in
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phosphatase activity of the mutant outer membrane fraction (Figure 2A). This was
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consistent with the observed location of the enzyme in the outer membrane fraction
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(Figure 1B). Complementation of Fn∆acpA with a plasmid containing the acpA gene
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restored outer membrane phosphatase activity to the wild-type level (Figure 2A). Using
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p-nitrophenyl palmitate as the substrate, we also measured an 8-fold decrease in
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phospholipase activity recovered from the outer membrane fraction of Fn∆acpA
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compared to FnWT (Figure 2B). These results are consistent with the predicted secretion
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of AcpA into the periplasmic space and suggest a tight association with the outer
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membrane. In addition, these data demonstrate that Fn∆acpA has significantly reduced
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enzymatic activities that are associated with AcpA.
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Fn∆acpA is defective in intramacrophage survival/replication
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The primary cellular target of F. tularensis during infection is the macrophage (26, 39).
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To examine the role that AcpA may play in protecting Francisella from phagocytic
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killing and intracellular trafficking, we measured survival of Fn∆acpA in the THP-1 cell
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line. While the FnWT and Fn∆acpA complemented strains replicated within the THP-1
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cells, the Fn∆AcpA mutant showed about a half-log reduction over the 12 hours post
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infection, with a slight recovery to the initial infection numbers by 24 hrs (Figure 3). The
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experiment was not continued beyond 24 hrs due to a loss in macrophage viability.
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Similar results were observed in murine and rat macrophage cell lines (data not shown).
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These results demonstrate that AcpA plays a role in intramacrophage survival and/or
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replication of FnWT.
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Electron microscopy demonstrates delayed phagosomal escape of the acpA mutant
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To further delineate the function of AcpA in intramacrophage survival, we used
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transmission electron microscopy to visualize PMA-stimulated THP-1 cells and MDMs
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infected with either FnWT or Fn∆acpA (Figures 4 & 5). At two hours post-infection in
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both macrophages, nearly all of the mutant and wild-type bacteria were contained in
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membrane-bound vacuoles, consistent with the known early compartmentalization of the
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pathogen in phagosomes (Figures 4A & 5A). After six hours, over half of FnWT had
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escaped from phagosomes (Figures 4B & 5B) and by 12 hours post infection, almost
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none of the wild-type pathogen was contained inside vacuoles with distinct membranes
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boundaries in THP-1 cells (Figures 4C). Studies with MDMs revealed similar findings,
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except that the escape of FnWT at 12 hours was slower, such that 40% were still in intact
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membranes (versus 75% of the Fn∆acpA, Figure 5C). In sharp contrast, we observed
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very little degradation of vacuoles containing Fn∆acpA during the 12-hour time frame of
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the experiments. After 12 hours, approximately 75 % of Fn∆acpA was still contained in
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vacuoles having distinct membrane borders (Figures 4D & 5D). These results suggest
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that AcpA plays a role in intramacrophage survival/replication via disruption of the
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phagosomal membrane that allows access of Francisella to the host cell cytosol.
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The Fn∆acpA mutant has reduced virulence in the mouse model
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To examine the role of AcpA in an animal model of infection, we measured survival rates
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of mice infected with FnWT and Fn∆acpA. After 36 hours following intraperitoneal
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infection, only 20 % of the mice infected with FnWT had survived, while 80 % of the
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mice infected with Fn∆acpA survived (Figure 6). At the 48-hour time point, all mice
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infected with FnWT were dead, while 60 % of those infected with Fn∆acpA survived.
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However by 72 hours post infection, all mice had died. Competition assays corroborated
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the apparent decrease in Fn∆acpA virulence, demonstrating a consistent competitive
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index of ~0.17 in both liver and spleen. These data indicate that loss of AcpA activity
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results in a less virulent pathogen, which is presumably due to decreased
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intramacrophage survival. Because mice infected with Fn∆acpA eventually died, it is
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likely that AcpA acts in concert with other factors to disrupt normal intracellular
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trafficking and that the influence of AcpA is largest in the initial stages of infection.
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Discussion
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Francisella AcpA possesses physical and chemical properties unlike other bacterial acid
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phosphatases (20, 31). This 57–kDa enzyme hydrolyzes a wide variety of physiologically
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meaningful substrates, including phosphorylated tyrosine peptides, inositol phosphates,
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phospholipid-like molecules, AMP, ATP, fructose 1,6-bisphosphate, glucose and fructose
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6-phosphosphates, NADP and ribose 5-phosphate, but fails to possess significant
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similarity to other acid phosphatases in the protein database (27-28). It has been recently
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crystallized (14) and the completed structure of this enzyme will likely provide important
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clues to its biological functions.
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Many acid phosphatases have been shown or predicted to play a role in virulence,
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most often in intracellular pathogens, by the inhibition of a respiratory burst. Such
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activity has been reported for AcpA of FnWT (27), as well as for Coxiella burnetii (4),
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Legionella spp. (31) and Leishmania (29). In this study, we described the role of
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Francisella AcpA, which has dual acid phosphatase and phospholipase C activities, in
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virulence and phagosomal trafficking in macrophages.
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In this work, we constructed a complete deletion of the acpA open reading frame.
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A previous study of AcpA in FnWT concluded that, upon the chromosomal deletion of a
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300 bp 3’ region and the replacement of this region by an erythromycin cassette, there
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was no effect upon intramacrophage survival or virulence (7). We were therefore
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somewhat surprised by the phenotypes observed due to the complete deletion of acpA. It
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is possible that, since the entire acpA orf was not deleted in the previous study, a
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functional truncated protein was still produced. With regard to acid phosphatase activity,
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this may be unlikely, as the peak of phosphatase activity in protein fractions associated
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with AcpA was absent in their mutant. However, it is possible that the truncated protein
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retained phospholipase activity, and it is this activity that is important in phagosomal
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trafficking.
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It was shown that the phosphatase and phospholipase activities of the Fn∆acpA
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mutant had 10-fold and 8-fold less activity than FnWT respectively, indicating that AcpA
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contributed to the overall activity but is not the only phosphatase or phospholipase in
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FnWT. Indeed, enzyme assays have shown at least two peaks of phosphatase activity in
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crude protein fractions (7) and genome scanning shows three other acid phosphatases in
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FnWT (unpublished observations). The future characterization and mutagenesis of these
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additional acid phosphatases will help to define the individual contributions of each
19
enzyme to the biology of Francisella.
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We determined the growth kinetics of the acpA mutant versus the FnWT in
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various macrophage cell lines where we showed that the acpA mutant exhibited a 10- to
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20-fold reduction in survival in THP-1 cells. This phenotype was due to the loss of acpA,
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as the complemented mutant strain revealed intramacrophage growth kinetics identical to
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the FnWT strain. To explore the survival or growth of the acpA mutant in vivo, mice were
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infected intraperitoneally (and intranasally with similar results, data not shown). We
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found that mice infected with the acpA mutant survived longer than mice infected with
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the FnWT strain and there was a corresponding reduction in organ bacterial load. When
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mice were infected with a mixture of FnWT and acpA mutant strains at a ratio of 1:1, the
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acpA mutant strain was less competitive than the FnWT strain (competitive index of
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~0.17 in both liver and spleen). These data suggest that AcpA contributes to virulence. It
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is possible that a greater virulence defect will be observed upon the deletion of multiple
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acid phosphatase genes.
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It has been shown that Francisella spp are initially contained within a phagosome
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but later escape into cytoplasm (11, 34-33). The factors involved in and the mechanism of
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phagosomal escape are largely unknown, but IglC and MglA have been shown to play a
13
role (33). We found that in THP-1 cells and MDMs, the Fn∆acpA mutant was primarily
14
still located inside the phagosome at 6 hrs post infection and only poorly escaped into the
15
cytoplasm after 12 hrs of infection, whereas, FnWT was nearly 100% and 60% escaped
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from phagosomes by 12 hrs of infection in THP-1s and MDMs, respectively. These data
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suggest that AcpA plays a role in intramacrophage trafficking, and the inability of the
18
mutant to escape from the phagosome likely contributes to its decreased intramacrophage
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survival and virulence.
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Because AcpA has numerous enzymatic targets, a challenge for the future is to
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determine which of these enzymatic activities underlies the observed respiratory burst
22
suppression and altered phagosomal escape due to AcpA. One possibility is that AcpA
23
hydrolyzes phospholipids of the phagosomal inner membrane, which might compromise
15
1
membrane integrity. Alternatively, AcpA might affect host signaling pathways by
2
dephosphorylation of host proteins, inositol phosphates or phosphoinositides, the latter
3
being critically important for phagosome formation (37) and respiratory burst activation
4
(5). A third hypothesis is that AcpA activity within the bacterium helps to induce or
5
amplify the activity of other proteins that are essential for phagosome disruption, such as
6
IglC and its regulator MglA (33, 22). In this context, the polyspecificity and high
7
abundance of AcpA may point to a phosphate scavenger role for the enzyme (41). Based
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on structural, enzymatic and phenotypic properties, inhibitors of AcpA and other acid
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phosphatases may be developed that, when used in combination with other antimicrobial
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agents, would provide alternative therapy against F. tularensis infection.
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Acknowledgements
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This work was supported by National Institutes of Health grants: U56AI057164
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(Midwest Center of Excellence (MWCE): Transmission/Pathogenesis of Bioterrorism
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Agents to LSS), U54AI057153 (Region V "Great Lakes" Regional Center of Excellence
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in Biodefense and Emerging Infectious Diseases Consortium (GLRCE) to LSS and JSG),
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U54AI057156 (Western Regional Center of Excellence for Biodefense and Emerging
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Infectious Diseases to JSG) and an NIH institutional training grant AI 0155411 (to AB).
19
We thank Dr. Karen Elkins and Fran Nano for providing guidance and Francisella
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strains, and Jack Tanner and Tom Reilly for their advice and critical reading of this
21
manuscript.
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1
Figure Legends
2
Figure 1. Construction and confirmation of the ∆acpA mutant. (A) Southern blot
3
analysis of the chromosomal DNA of the ∆acpA mutant vs. the FnWT. DNAs were
4
digested with EcoRI and probed with the acpA orf (lanes 1 and 2) or the kan cassette
5
(lanes 3 and 4) showing the expected differential hybridization of the probe. An EcoRI
6
site within the acpA orf results in two hybridizing bands, while no EcoRI sites are within
7
the acpA orf. Lane 1 and 3, F. novicida; lane 2 and 4, F. novicida ∆acpA. (B) Western
8
blot analysis of whole cell lysates (WC), outer membrane fractions (OM) and inner
9
membrane fractions (IM) of FnWT, Fn∆acpA, and Schu 4 detecting the 57 kDa AcpA
10
D E
T P
E C
protein. Rabbit polyclonal anti-AcpA sera was used as the primary antibody.
11 12
Figure 2. Acid phosphatase (A) and lipase activity (B) of inner membrane (IM), outer
13
membrane (OM) and cytoplasmic (Cyt) fractions of Fn∆acpA (gray bars) versus the
14
Fn∆acpA complemented (white bars) and parental strains (black bars). Data represents
15
the mean +/- the standard deviation of three independent experiments each with duplicate
16
wells.
17
C A
18
Figure 3. Intramacrophage survival assays performed in THP-1 human macrophage-like
19
cell lines with Fn∆acpA (square), ∆acpA complemented (triangle) and parental strains
20
(diamond). Data represents the mean +/- the standard deviation of three independent
21
experiments each with duplicate wells for each time point. Asterisks denote significant
22
differences of the ∆acpA mutant vs. the FnWTat 12 and 24 hrs. post-infection (p
