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Progression of pneumonic infection. Mice were infected with 5610 4-1610 5 CFU of Y. pestis Lux PcysZK intranasally and imaged using an IVIS Spectrum. (A) Sequential images from representative animals. (B) For each animal, average bioluminescence was calculated for the thoracic cavity using the ROI tool in Living Image 3.2 software package. Black and white symbols represent animals infected with WT or Dpla Y. pestis, respectively. Dotted line represents the limit of detection based on images from uninfected animals. ** = p,0.005, *** = p,0.001. At various time points, lungs were harvested from a subset of animals to determine bacterial loads (CFU) and compared to bioluminescence from the thoracic cavity (C) or from the lungs ex vivo (D). doi:10.1371/journal.pone.0047123.g008
Source publication
Yersinia pestis causes an acute infection known as the plague. Conventional techniques to enumerate Y. pestis can be labor intensive and do not lend themselves to high throughput assays. In contrast, bioluminescent bioreporters produce light that can be detected using plate readers or optical imaging platforms to monitor bacterial populations as a...
Contexts in source publication
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... can be used to monitor pneumonic infection, we challenged mice intranasally with the WT CO92 Lux PcysZK strain and followed the progression of pneumonic plague by optical imaging. Bioluminescent signal could be detected from the thoracic cavity of all mice as early as 24 hrs post-inoculation and increased throughout the course of infection (Fig. 8A and B). To demonstrate that the bioluminescence signal directly correlated with bacterial numbers, lungs were harvested from a subset of animals after the 24, 48, and 72 hrs imaging sessions. The tissues were imaged and bacterial numbers in the lungs were determined. Bioluminescent signal from imaging of the thoracic cavity directly ...
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... demonstrate that the bioluminescence signal directly correlated with bacterial numbers, lungs were harvested from a subset of animals after the 24, 48, and 72 hrs imaging sessions. The tissues were imaged and bacterial numbers in the lungs were determined. Bioluminescent signal from imaging of the thoracic cavity directly correlated to lung CFU ( Fig. 8C; R 2 = 0.8323). The significance of the correlation increased further when comparing signal directly from harvested lungs to CFU ( Fig. 8D; R 2 = 0.9684). Animals infected with the Lux PcysZK strain succumbed to infection between 60 and 80 hrs post-infection, a similar time to death as seen for Y. pestis without a reporter ...
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... the 24, 48, and 72 hrs imaging sessions. The tissues were imaged and bacterial numbers in the lungs were determined. Bioluminescent signal from imaging of the thoracic cavity directly correlated to lung CFU ( Fig. 8C; R 2 = 0.8323). The significance of the correlation increased further when comparing signal directly from harvested lungs to CFU ( Fig. 8D; R 2 = 0.9684). Animals infected with the Lux PcysZK strain succumbed to infection between 60 and 80 hrs post-infection, a similar time to death as seen for Y. pestis without a reporter ...
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... the model for pneumonic plague, the Dpla mutant colonizes the lungs but is unable to proliferate in these tissues [27]. As expected, we observed low levels of bioluminescence from the thoracic cavity of mice infected intranasally with Y. pestis Dpla Lux PcysZK , correlating with low levels of bacteria in these tissues (Fig. 8). Importantly, compared to WT infected mice, lumines- cence from the Dpla infected animals was significantly lower at all time points, except at the 72 hr time point when there were not enough WT animals to calculate significance (Fig. 8B). While the Dpla mutant does not proliferate within the lungs during pneumonic infection, the LD 50 ...
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... intranasally with Y. pestis Dpla Lux PcysZK , correlating with low levels of bacteria in these tissues (Fig. 8). Importantly, compared to WT infected mice, lumines- cence from the Dpla infected animals was significantly lower at all time points, except at the 72 hr time point when there were not enough WT animals to calculate significance (Fig. 8B). While the Dpla mutant does not proliferate within the lungs during pneumonic infection, the LD 50 of the mutant is similar to WT Y. pestis, likely due to the development of septicemic plague [27]. The sensitivity of the Lux PcysZK bioreporter allowed us to observe the development of septicemic plague in Dpla infected animals (Fig. ...
Citations
... 11 These strains also harbored bioluminescent bioreporters (pLUX or Lux ptolC ) to monitor bacterial viability as a function of bioluminescence. 12,13 These strains represent a surrogate for fully virulent Y. pestis that can be handled safely at BSL-2 to develop and validate inactivation procedures. 8,11 For in vivo studies performed within the BSL-3, we used a fully virulent strain of Y. pestis KIM5+ which harbors both the pgm locus and pCD1. ...
... 15 C57BL/6J mice were anesthetized with ketamine/xylazine and administered 20 lL of fully virulent Y. pestis suspended in 1 · Dulbecco's PBS to the left nare as previously described. 13,15 At 48 h postinfection, lungs, spleen, and liver were removed by sterile necropsy. Tissues from five mice were cut in half, prior to adding to tissue cassettes, and submerged in 10% NBF for 24 h, 12 mL per tissue. ...
... C57BL/6J mice were anesthetized with ketamine/ xylazine and administered 20 lL of fully virulent Y. pestis as previously described. 13,15 At 6, 12, 24, 36, and 48 h postinfection, whole lungs were necropsied and homogenized as described above. Bacterial CFU were enumerated from each sample by serial dilution of 100 lL of the homogenized samples to determine the bacterial concentration prior to inactivation. ...
... Furthermore, independent experiments with a Y. pestis strain with a luciferase bioreporter (Y. pestis CO92 Lux pcysZK ), which allows for monitoring bacterial proliferation via optical imaging and host survival in the same group [48], showed no significant differences between the two mouse lines in bacterial proliferation at later time points or in the mean-time to death (Fig 2B and 2C). These data indicate that the loss of LTB 4 -BLT1 signaling in BLT1 -/mice does not impact the infectivity of Y. pestis, further supporting that LTB 4 synthesis and signaling is disrupted during pneumonic plague. ...
... 6-8 week-old C57BL/ 6J or BLT1 -/- [49] male and female mice were infected with Y. pestis KIM5+ or Y. pestis CO92 LUX pcysZK . For lipid measurements, mice were anesthetized with ketamine/xylazine and administered 20 μL of Y. pestis KIM5+ suspended in 1× DPBS to the left nare as previously described [48,100]. Mice were monitored for the development of moribund disease symptoms twice daily and humanely euthanized when they met previously approved end point criteria. ...
... At each time point, mice were anesthetized with isoflurane and imaged using the IVIS Spectrum imaging system (Caliper Life Sciences, Hopkinton, MA). Average radiance (photons/s/cm2) was calculated for the lungs as previously described [48]. For the exogenous LTB 4 treatment, mice were intraperitoneally injected with 1 x DPBS or 10 nmol LTB 4 (Cayman Chemical Cat. ...
Subverting the host immune response to inhibit inflammation is a key virulence strategy of Yersinia pestis . The inflammatory cascade is tightly controlled via the sequential action of lipid and protein mediators of inflammation. Because delayed inflammation is essential for Y . pestis to cause lethal infection, defining the Y . pestis mechanisms to manipulate the inflammatory cascade is necessary to understand this pathogen’s virulence. While previous studies have established that Y . pestis actively inhibits the expression of host proteins that mediate inflammation, there is currently a gap in our understanding of the inflammatory lipid mediator response during plague. Here we used the murine model to define the kinetics of the synthesis of leukotriene B4 (LTB 4 ), a pro-inflammatory lipid chemoattractant and immune cell activator, within the lungs during pneumonic plague. Furthermore, we demonstrated that exogenous administration of LTB 4 prior to infection limited bacterial proliferation, suggesting that the absence of LTB 4 synthesis during plague contributes to Y . pestis immune evasion. Using primary leukocytes from mice and humans further revealed that Y . pestis actively inhibits the synthesis of LTB 4 . Finally, using Y . pestis mutants in the Ysc type 3 secretion system (T3SS) and Yersinia outer protein (Yop) effectors, we demonstrate that leukocytes recognize the T3SS to initiate the rapid synthesis of LTB 4 . However, several Yop effectors secreted through the T3SS effectively inhibit this host response. Together, these data demonstrate that Y . pestis actively inhibits the synthesis of the inflammatory lipid LTB 4 contributing to the delay in the inflammatory cascade required for rapid recruitment of leukocytes to sites of infection.
... 8,13,36 To determine if MNA inoculation results in a similar disease manifestation, MNAs were used to inoculate mice with a Y. pestis strain expressing a bioluminescent bioreporter. 37 iScience Article has been reported for intradermal needle inoculation or flea transmission, 8,36 supporting that MNA inoculation is a viable mechanism to model intradermal infections by Y. pestis. ...
... pestis znuA ybtX) expressing a bioluminescent bioreporter. 37 A subset of mice was euthanized at 15 min post-inoculation to confirm that equivalent iScience Article inoculums were delivered (750 G 117 and 526 G 183, respectively), and bacterial proliferation and dissemination were monitored in the remaining mice by optical imaging. Greater bioluminescence was detected at each time point in the ears of S100A9 À/À mice compared to C57BL6/J mice infected with Y. pestis ( Figures 3A and 3B), suggesting that calprotectin restricts bacterial proliferation within the dermis. ...
... Detailed methods are provided in the online version of this paper and include the following: 50 and Y. pestis znuA ybtX 51 were routinely grown in Difco brain heart infusion (BHI) broth (BD Biosciences). Strains transformed with the pGEN-luxCDABE or pGEN222-dsRed plasmids 37 were grown in the presence of kanamycin (50 mg/mL). For infection, bacteria were grown overnight at 26 C in BHI broth prior to intradermal challenge. ...
Fleas transmit Yersinia pestis directly within the dermis of mammals to cause bubonic plague. Syringe-mediated inoculation is widely used to recapitulate bubonic plague and study Y. pestis pathogenesis. However, intradermal needle inoculation is tedious, error prone, and poses a significant safety risk for laboratorians. Microneedle arrays (MNAs) are micron-scale polymeric structures that deliver materials to the dermis, while minimizing the risk of needle sticks. We demonstrated that MNA inoculation is a viable strategy to recapitulate bubonic plague and study bacterial virulence by defining the parameters needed to establish a lethal infection in the mouse model and characterizing the course of infection using live-animal optical imaging. Using MNAs, we also demonstrated that Y. pestis must overcome calprotectin-mediated zinc restriction within the dermis and dermal delivery of an attenuated mutant has vaccine potential. Together, these data demonstrate that MNAs are a safe alternative to study Y. pestis pathogenesis in the laboratory.
... In vivo calprotectin measurements were made from animals infected with Y. pestis CO92 [pCD1 (+) pMT (+) pPCP1 (+) pgm (+) ]. For in vitro calprotectin experiments, strains were transformed with the pGEN-luxCDABE plasmid (104) to monitor bacterial growth by bioluminescence, as we have previously described (63), and were grown in the presence of carbenicillin (50 mg/mL). Y. pestis was routinely grown for 15 to 18 h at 26°C in Difco BHI broth (BD Biosciences) with aeration. ...
... Animal Infections with Y. pestis. Mice were challenged with Y. pestis as previously described (63,105,106). Briefly, for intranasal challenge, mice were anesthetized with ketamine/xylazene and administered 20 mL bacteria suspended in 1× PBS to the left nare. ...
... Around 1 × 10 5 CFU were transferred to individual wells of a white 96-well plate (Greiner Bio-One) containing calprotectin in 38% BHI and 62% calprotectin buffer (20 mM Tris, pH 7.5, 100 mM NaCl, and 3 mM CaCl 2 ). Plates were incubated at 37°C, and bacterial growth as a function of bioluminescence was measured using a Biotek Synergy HT plate reader (0.5 s read, sensitivity of 135), as previously described (63). ...
Significance
Transition metals are required for proper cellular function, which renders them critical for all life. To restrict bacterial infection, eukaryotic organisms actively sequester these transition metals, a concept referred to as nutritional immunity. Consequently, bacterial pathogens have evolved dedicated mechanisms to acquire transition metals in order to colonize the host. During human plague, Yersinia pestis overcomes iron limitation via the production of the secreted siderophore yersiniabactin. Here, we identify an iron-independent role for yersiniabactin in evading zinc-mediated nutritional immunity during mammalian infection and in Y. pestis colonization of the flea–insect vector. Importantly, yersiniabactin is found in several pathogens, indicating that a variety of bacteria use it to acquire multiple metals in order to overcome nutritional immunity.
... The growth in M9, LB, and BHI was monitored by measuring the OD 600nm , whereas growth in blood and its derivatives was measured by counting the colony-forming units (CFUs), as described previously [12]. Lastly, growth in flea gut content was determined by measuring bioluminescence [29]. ...
... The strain was built as follows. First, we inserted the miniTn7-aphA-P tolC ::lux at the att Tn7 site by co-transforming Y. pestis with pLOU034 and pTNS2 [29,30]. Next, we (i) deleted the aphA antibiotic resistance cassette flanked by flippase recognition target (FRT) sites using the pFLP2 plasmid [30], (ii) cured the pFLP2 by streaking bacteria on plates of LB agar supplemented with 5% sucrose, and (iii) replaced the tolC promoter with a kanamycin resistance cassette by applying the lambda Red recombinase system and the plasmids and primer sets listed in the Supplementary Tables [16]. ...
... A new in vivo method for rapid identifying Y. pestis genes required for flea blockage We sought to develop a means of circumventing the following obstacle: the flea infection model currently used to identify Y. pestis genes required for blockage (by screening libraries of Y. pestis mutants) is unsuited to high-throughput screening. To this end, we took advantage of the properties of the luxCDABE operon from Photorhabdus luminescens because it confers bacteria with the ability to produce light autonomously and has been used as a bioreporter for monitoring bacteria in live animal models of infection [29,[33][34][35]. We first attempted to generate a Y. pestis strain with the brightest, most innocuous bioreporter construct, so that it could be used to identify the bacterial genes needed for flea blockage. ...
To thrive, vector-borne pathogens must survive in the vector’s gut. How these pathogens successfully exploit this environment in time and space has not been extensively characterized. Using Yersinia pestis (the plague bacillus) and its flea vector, we developed a bioluminescence-based approach and employed it to investigate the mechanisms of pathogenesis at an unprecedented level of detail. Remarkably, lipoylation of metabolic enzymes, via the biosynthesis and salvage of lipoate, increases the Y. pestis transmission rate by fleas. Interestingly, the salvage pathway’s lipoate/octanoate ligase LplA enhances the first step in lipoate biosynthesis during foregut colonization but not during midgut colonization. Lastly, Y. pestis primarily uses lipoate provided by digestive proteolysis (presumably as lipoyl peptides) rather than free lipoate in blood, which is quickly depleted by the vector. Thus, spatial and temporal factors dictate the bacterium’s lipoylation strategies during an infection, and replenishment of lipoate by digestive proteolysis in the vector might constitute an Achilles’ heel that is exploited by pathogens.
... Three previous studies have followed the progression of bubonic plague in mice following intradermal [27,28] or subcutaneous [29] injection of bioluminescent Y. pestis. Our results using the natural flea-borne route of transmission show some differences to these studies. ...
Yersinia pestis can be transmitted by fleas during the first week after an infectious blood meal, termed early-phase or mass transmission, and again after Y . pestis forms a cohesive biofilm in the flea foregut that blocks normal blood feeding. We compared the transmission efficiency and the progression of infection after transmission by Oropsylla montana fleas at both stages. Fleas were allowed to feed on mice three days after an infectious blood meal to evaluate early-phase transmission, or after they had developed complete proventricular blockage. Transmission was variable and rather inefficient by both modes, and the odds of early-phase transmission was positively associated with the number of infected fleas that fed. Disease progression in individual mice bitten by fleas infected with a bioluminescent strain of Y . pestis was tracked. An early prominent focus of infection at the intradermal flea bite site and dissemination to the draining lymph node(s) soon thereafter were common features, but unlike what has been observed in intradermal injection models, this did not invariably lead to further systemic spread and terminal disease. Several of these mice resolved the infection without progression to terminal sepsis and developed an immune response to Y . pestis , particularly those that received an intermediate number of early-phase flea bites. Furthermore, two distinct types of terminal disease were noted: the stereotypical rapid onset terminal disease within four days, or a prolonged onset preceded by an extended, fluctuating infection of the lymph nodes before eventual systemic dissemination. For both modes of transmission, bubonic plague rather than primary septicemic plague was the predominant disease outcome. The results will help to inform mathematical models of flea-borne plague dynamics used to predict the relative contribution of the two transmission modes to epizootic outbreaks that erupt periodically from the normal enzootic background state.
... 7 Compared with a chromosome-based reporter, the plasmidbased lux reporter could offer highresolution and high-definition imaging of the pathophysiological processes of infection because it can be maintained at higher copies, which can increase the sensitivity of bioluminescent signals. 8 In this current study, a well-developed strain of Gram-negative bacteria, E. coli O157:H7-lux, expressing bacterial luciferase was used to perform intranasal infection in mice 4 days post-influenza A virus/Puerto Rico/8 (IAV/PR8) infection. Using bioluminescence imaging, the colonization and clearance process of E. coli O157:H7-lux in the lung following IAV/PR8 infection was monitored in this mouse model. ...
Objective
To develop a bioluminescence-labelled bacterial infection model to monitor the colonization and clearance process of Escherichia coli O157:H7 in the lungs of mice following influenza A virus/Puerto Rico/8/34 (H1N1) strain (IAV/PR8) infection.
Methods
BALB/c mice were administered IAV/PR8 or 0.01 M phosphate-buffered saline (PBS; pH 7.4) intranasally 4 days prior to intranasal administration of 1 × 10⁷ colony-forming units (CFU) of E. coli O157:H7-lux. Whole-body bioluminescent signals were monitored at 10 min, 4 h, 8 h, 12 h, 16 h and 24 h post-bacterial infection. Lung bioluminescent signals and bacterial load (CFU/g) were monitored at 4 h, 8 h, 12 h, 16 h and 24 h post-bacterial infection.
Results
Prior IAV/PR8 infection of mice resulted in a higher level of bacterial colonization and a lower rate of bacterial clearance from the lungs compared with mice treated with PBS. There were also consistent findings between the bioluminescence imaging and the CFU measurements in terms of identifying bacterial colonization and monitoring the clearance dynamics of E. coli O157:H7-lux in mouse lungs.
Conclusion
This novel bioluminescence-labelled bacterial infection model rapidly detected bacterial colonization of the lungs and monitored the clearance dynamics of E. coli O157:H7-lux following IAV/PR8 infection.
... Using a combination of siRNAs targeting genes of variable expression levels, we optimized Lipofectamine/siRNA concentrations and transfection time to consistently achieve Ͼ70% knockdown of targets ( Fig. 1A and B). Next, we infected cells transfected with Rab2 siRNA and Cop1 siRNA (both siRNAs are known to inhibit intracellular survival of other pathogens [41,42,48]) with Y. pestis CO92 pCD1 (-) Lux PtolC , a bioluminescent bioreporter that can differentiate as little as a twofold difference in intracellular bacteria ( Fig. 1C and D; R 2 ϭ 0.89) (50), to demonstrate that this bioreporter can be used to kinetically monitor changes in intracellular survival (Fig. 1E). Finally, using Cop1 siRNA as a positive control, we calculated Z= factor values for this assay of 0.61 and 0.83 at 2 and 10 h postinfection, respectively (Fig. 1F). ...
... Furthermore, the primary hits were validated against two different Y. pestis strains, one with the Ysc type three secretion system (T3SS) (KIMD19 pCD1 (ϩ) Lux PtolC ) and one without it (CO92 pCD1 (-) Lux PtolC ). A direct correlation was observed between the two strains ( Fig. 2B; r s ϭ 0.87), supporting previous studies showing that the T3SS is dispensable for intracellular survival (16,24,35,50). From the primary hits, 135 genes showed Ն40% inhibition of intracellular survival of Y. pestis and 7 showed a hypervirulent phenotype with Ն20% more growth than scrambled controls. ...
... E. coli K-12 DH5␣ (New England Biolabs) and Salmonella enterica Typhimurium L2 (ATCC 14028s) were cultivated at 37°C in Luria-Bertani broth (Miller) (Becton, Dickinson, and Co.). Bioluminescent derivatives were generated using the Lux PtolC bioreporter as described previously (50). To generate fluorescent bacterial strains, bacteria were transformed with pGEN222, pGEN-P EM7 ::DsRED, or pGEN222::mCherry (72) and maintained with 50 g/ml carbenicillin for 30 min at room temperature as previously described (35). ...
Yersinia pestis has evolved many strategies to evade the innate immune system. One of these strategies is the ability to survive within macrophages. Upon phagocytosis, Y. pestis prevents phagolysosome maturation and establishes a modified compartment termed the Yersinia-containing vacuole (YCV). Y. pestis actively inhibits the acidification of this compartment, and eventually, the YCV transitions from a tight-fitting vacuole into a spacious replicative vacuole. The mechanisms to generate the YCV have not been defined. However, we hypothesized that YCV biogenesis requires Y. pestis interactions with specific host factors to subvert normal vesicular trafficking. In order to identify these factors, we performed a genome-wide RNA interference (RNAi) screen to identify host factors required for Y. pestis survival in macrophages. This screen revealed that 71 host proteins are required for intracellular survival of Y. pestis. Of particular interest was the enrichment for genes involved in endosome recycling. Moreover, we demonstrated that Y. pestis actively recruits Rab4a and Rab11b to the YCV in a type three secretion system-independent manner, indicating remodeling of the YCV by Y. pestis to resemble a recycling endosome. While recruitment of Rab4a was necessary to inhibit YCV acidification and lysosomal fusion early during infection, Rab11b appeared to contribute to later stages of YCV biogenesis. We also discovered that Y. pestis disrupts global host endocytic recycling in macrophages, possibly through sequestration of Rab11b, and this process is required for bacterial replication. These data provide the first evidence that Y. pestis targets the host endocytic recycling pathway to avoid phagolysosomal maturation and generate the YCV.
... Bacteria were propagated in Difco Brain Heart Infusion (BHI) (BD, Co.). We have previously shown that the bioluminescence generated by the Lux PtolC bioreporter directly correlates with bacterial numbers and can be used to kinetically monitor Y. pestis intracellular survival in gentamicin protection assays (Sun et al., 2012;Connor et al., 2015). ...
... Concentrations of gentamicin used were not cytotoxic to macrophages. Bacterial numbers, as a function of bioluminescence produced by the Lux PtolC bioreporter (Sun et al., 2012;Connor et al., 2015), were monitored kinetically to limit manipulation of the macrophages using a Synergy HT plate reader (0.5 s read, sensitivity of 135) (BioTek) or IVIS Spectrum camera system (5 sec with medium binning through a 500 nm emission filter) (Caliper). ...
... Gentamicin was diluted in 37 • C DMEM + 10% FBS and added to the bacteria to achieve the final desired concentration (0-128 µg/ml). One h after treatment, bacterial numbers were determined by conventional enumeration using serial dilution and plating on BHI agar (Sun et al., 2012). ...
The study of intracellular bacterial pathogens in cell culture hinges on inhibiting extracellular growth of the bacteria in cell culture media. Aminoglycosides, like gentamicin, were originally thought to poorly penetrate eukaryotic cells, and thus, while inhibiting extracellular bacteria, these antibiotics had limited effect on inhibiting the growth of intracellular bacteria. This property led to the development of the antibiotic protection assay to study intracellular pathogens in vitro. More recent studies have demonstrated that aminoglycosides slowly penetrate eukaryotic cells and can even reach intracellular concentrations that inhibit intracellular bacteria. Therefore, important considerations, such as antibiotic concentration, incubation time, and cell type need to be made when designing the antibiotic protection assay to avoid potential false positive/negative observations. Yersinia pestis, which causes the human disease known as the plague, is a facultative intracellular pathogen that can infect and replicate in macrophages. Y. pestis is sensitive to gentamicin and this antibiotic is often employed in the antibiotic protection assay to study the Y. pestis intracellular life cycle. However, a large variety of gentamicin concentrations and incubation periods have been reported in the Y. pestis literature without a clear characterization of the potential influences that variations in the gentamicin protection assay could have on intracellular growth of this pathogen. This raised concerns that variations in the gentamicin protection assay could influence phenotypes and reproducibility of data. To provide a better understanding of the potential consequences that variations in the gentamicin protection assay could have on Y. pestis, we systematically examined the impact of multiple variables of the gentamicin protection assay on Y. pestis intracellular survival in macrophages. We found that prolonged incubation periods with low concentrations of gentamicin, or short incubation periods with higher concentrations of the antibiotic, have a dramatic impact on intracellular growth. Furthermore, the degree of sensitivity of intracellular Y. pestis to gentamicin was also cell type dependent. These data highlight the importance to empirically establish cell type specific gentamicin protection assays to avoid potential artificial data in Y. pestis intracellular studies.
... Intracellular bacterial numbers were determined by conventional CFU enumeration in triplicate as previously described. 70 Three independent trials with RAW274.6 cells were performed; one representative trial is shown. ...
A number of bacterial pathogens require the ZnuABC Zinc (Zn2+) transporter and/or a second Zn2+ transport system to overcome Zn2+ sequestration by mammalian hosts. Previously we have shown that in addition to ZnuABC, Yersinia pestis possesses a second Zn2+ transporter that involves components of the yersiniabactin (Ybt), siderophore-dependent iron transport system. Synthesis of the Ybt siderophore and YbtX, a member of the major facilitator superfamily, are both critical components of the second Zn2+ transport system. Here we demonstrate that a ybtX znu double mutant is essentially avirulent in mouse models of bubonic and pneumonic plague while a ybtX mutant retains high virulence in both plague models. While sequestration of host Zn is a key nutritional immunity factor, excess Zn appears to have a significant antimicrobial role in controlling intracellular bacterial survival. Here, we demonstrate that ZntA, a Zn2+ exporter, plays a role in resistance to Zn toxicity in vitro, but that a zntA zur double mutant retains high virulence in both pneumonic and bubonic plague models and survival in macrophages. We also confirm that Ybt does not directly bind Zn2+ in vitro under the conditions tested. However, we detect a significant increase in Zn2+-binding ability of filtered supernatants from a Ybt+ strain compared to those from a strain unable to produce the siderophore, supporting our previously published data that Ybt biosynthetic genes are involved in the production of a secreted Zn-binding molecule (zincophore). Our data suggest that Ybt or a modified Ybt participate in or promote Zn-binding activity in culture supernatants and is involved in Zn acquisition in Y. pestis.
