Further characterization of a highly attenuated Yersinia pestis CO92 mutant deleted for the genes encoding Braun lipoprotein and plasminogen activator protease in murine alveolar and primary human macrophages

Lier, Christina J. van; Tiner, Bethany L.; Chauhan, Sadhana; Motin, Vladimir L.; Fitts, Eric C.; Huante, Matthew B.; Endsley, Janice J.; Ponnusamy, Duraisamy; Sha, Jian; Chopra, Ashok K.

doi:10.1016/j.micpath.2015.02.005

Cited by 12 publications

(9 citation statements)

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“…While intracellular vs. extracellular bacteria can be differentiated during live imaging, in vitro assays that do not use microscopy are not able to differentiate between the two, thus requiring the use of an antibiotic protection assay. However, as a wide variety of concentrations of gentamicin have been reported in the Yersinia literature (Pujol and Bliska, 2003 ; Benedek et al, 2004 ; Leigh et al, 2005 ; Ponnusamy et al, 2011 ; Sha et al, 2013 ; Spinner et al, 2014 ; Tiner et al, 2015 ; van Lier et al, 2015 ), we next wanted to determine if extended incubation with gentamicin could influence intracellular proliferation of Y. pestis .…”

Section: Resultsmentioning

confidence: 99%

“…In most cases gentamicin is the primary antibiotic employed for Y. pestis intracellular studies because the bacterium is sensitive to the antibiotic (MIC ~2 μg/ml) (Smith et al, 1995 ). Surprisingly, in light of the potential influence gentamicin has on the intracellular growth of other bacteria, a large variety of gentamicin concentrations (ranging from 0.016 to 256 μg/ml) and incubation times (from 15 min to 2 h) have been reported in the Y. pestis literature (Pujol and Bliska, 2003 ; Benedek et al, 2004 ; Leigh et al, 2005 ; Ponnusamy et al, 2011 ; Sha et al, 2013 ; Spinner et al, 2014 ; Tiner et al, 2015 ; van Lier et al, 2015 ). Not surprisingly, variation in Y. pestis intracellular survival has also been observed.…”

Section: Introductionmentioning

confidence: 99%

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Impact of Gentamicin Concentration and Exposure Time on Intracellular Yersinia pestis

VanCleave

Pulsifer

Connor

et al. 2017

Front. Cell. Infect. Microbiol.

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Impact of Gentamicin Concentration and Exposure Time on Intracellular Yersinia pestis

VanCleave

Pulsifer

Connor

et al. 2017

Front. Cell. Infect. Microbiol.

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“…The samples were incubated at 37°C for 2 h with shaking at 180 rpm. The number of surviving bacteria (the number of CFU) in each sample was determined by serial dilution and plating on SBA plates (59)(60)(61). Percent bacterial survival was calculated by dividing the average number of CFU in PBS by the average number of CFU in each serum sample.…”

Section: Methodsmentioning

confidence: 99%

Combating Multidrug-Resistant Pathogens with Host-Directed Nonantibiotic Therapeutics

Andersson

Sha

Kirtley

et al. 2018

Antimicrob Agents Chemother

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Earlier, we reported that three Food and Drug Administration-approved drugs, trifluoperazine (TFP; an antipsychotic), amoxapine (AXPN; an antidepressant), and doxapram (DXP; a breathing stimulant), identified from an murine macrophage cytotoxicity screen, provided mice with 40 to 60% protection against pneumonic plague when administered at the time of infection for 1 to 3 days. In the present study, the therapeutic potential of these drugs against pneumonic plague in mice was further evaluated when they were administered at up to 48 h postinfection. While the efficacy of TFP was somewhat diminished as treatment was delayed to 24 h, the protection of mice with AXPN and DXP increased as treatment was progressively delayed to 24 h. At 48 h postinfection, these drugs provided the animals with significant protection (up to 100%) against challenge with the agent of pneumonic or bubonic plague when they were administered in combination with levofloxacin. Likewise, when they were used in combination with vancomycin, all three drugs provided mice with 80 to 100% protection from fatal oral infection when they were administered at 24 h postinfection. Furthermore, AXPN provided 40 to 60% protection against respiratory infection with when it was administered at the time of infection or at 24 h postinfection. Using the same cytotoxicity assay, we identified an additional 76/780 nonantibiotic drugs effective against For, 121 nonantibiotic drugs were identified to inhibit bacterium-induced cytotoxicity in murine macrophages. Of these 121 drugs, 13 inhibited the macrophage cytotoxicity induced by two additional multiple-antibiotic-resistant strains. Six of these drugs decreased the intracellular survival of all three strains in macrophages. These results provided further evidence of the broad applicability and utilization of drug repurposing screening to identify new therapeutics to combat multidrug-resistant pathogens of public health concern.

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“…Twofold serially diluted serum was then added to the wells of the ELISA microtiter plates, followed by the addition of secondary horseradish peroxidase (HRP)-conjugated anti-mouse specific antibodies to IgG, its isotypes, and/or IgA. The ELISA was performed as we described previously (28).…”

mentioning

confidence: 99%

A Replication-Defective Human Type 5 Adenovirus-Based Trivalent Vaccine Confers Complete Protection against Plague in Mice and Nonhuman Primates

Sha

Kirtley

Klages

et al. 2016

Clin Vaccine Immunol

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j Currently, no plague vaccine exists in the United States for human use. The capsular antigen (Caf1 or F1) and two type 3 secretion system (T3SS) components, the low-calcium-response V antigen (LcrV) and the needle protein YscF, represent protective antigens of Yersinia pestis. We used a replication-defective human type 5 adenovirus (Ad5) vector and constructed recombinant monovalent and trivalent vaccines (rAd5-LcrV and rAd5-YFV) that expressed either the codon-optimized lcrV or the fusion gene designated YFV (consisting of ycsF, caf1, and lcrV). Immunization of mice with the trivalent rAd5-YFV vaccine by either the intramuscular (i.m.) or the intranasal (i.n.) route provided protection superior to that with the monovalent rAd5-LcrV vaccine against bubonic and pneumonic plague when animals were challenged with Y. pestis CO92. Preexisting adenoviral immunity did not diminish the protective response, and the protection was always higher when mice were administered one i.n. dose of the trivalent vaccine (priming) followed by a single i.m. booster dose of the purified YFV antigen. Immunization of cynomolgus macaques with the trivalent rAd5-YFV vaccine by the prime-boost strategy provided 100% protection against a stringent aerosol challenge dose of CO92 to animals that had preexisting adenoviral immunity. The vaccinated and challenged macaques had no signs of disease, and the invading pathogen rapidly cleared with no histopathological lesions. This is the first report showing the efficacy of an adenovirus-vectored trivalent vaccine against pneumonic plague in mouse and nonhuman primate (NHP) models. Yersinia pestis is the causative agent of plague and can be transmitted to humans via an infected flea bite or by direct inhalation of the aerosolized bacilli from an infected person or an animal (1, 2). Plague manifests itself in three major forms in humans, namely, bubonic, septicemic, and pneumonic (2). Pneumonic plague is the most feared form due to its rapid onset and associated high mortality rate (1, 2). Y. pestis has been responsible for at least three pandemics in the past, which killed more than 200 million people (3). Current epidemiological records suggest that there are 4,000 human plague cases annually worldwide (2). The emergence of multi-antibiotic-resistant Y. pestis strains from plague patients and the potential of malicious dissemination of recombinantly engineered bacteria as an airborne bioweapon necessitate the development of an effective preexposure and/or postexposure prophylaxis treatment (1, 2).Currently, no Food and Drug Administration (FDA)-licensed plague vaccine exists in the United States, and recent efforts have focused on the development of recombinant subunit plague vaccines consisting of two well-characterized Y. pestis antigens, the F1 capsular antigen and the type 3 secretion system (T3SS) component and effector LcrV (4-8). F1, encoded by the caf1 gene, has a polymeric structure and confers bacterial resistance to phagocytosis (9). The F1-V-based vaccines are generally protective ag...

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Further characterization of a highly attenuated Yersinia pestis CO92 mutant deleted for the genes encoding Braun lipoprotein and plasminogen activator protease in murine alveolar and primary human macrophages

Cited by 12 publications

References 61 publications

Impact of Gentamicin Concentration and Exposure Time on Intracellular Yersinia pestis

Impact of Gentamicin Concentration and Exposure Time on Intracellular Yersinia pestis

Combating Multidrug-Resistant Pathogens with Host-Directed Nonantibiotic Therapeutics

A Replication-Defective Human Type 5 Adenovirus-Based Trivalent Vaccine Confers Complete Protection against Plague in Mice and Nonhuman Primates

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