Novel Genetic Tools for Diaminopimelic Acid Selection in Virulence Studies of Yersinia pestis

Molecular studies of bacterial virulence are enhanced by expression of recombinant DNA during infection to allow complementation of mutants and expression of reporter proteins in vivo. For highly pathogenic bacteria, such as Yersinia pestis, these studies are currently limited because deliberate introduction of antibiotic resistance is restricted to those few which are not human treatment options. In this work, we report the development of alternatives to antibiotics as tools for host-pathogen research during Yersinia pestis infections focusing on the diaminopimelic acid (DAP) pathway, a requirement for cell wall synthesis in eubacteria. We generated a mutation in the dapA-nlpB(dapX) operon of Yersinia pestis KIM D27 and CO92 which eliminated the expression of both genes. The resulting strains were auxotrophic for diaminopimelic acid and this phenotype was complemented in trans by expressing dapA in single and multi-copy. In vivo, we found that plasmids derived from the p15a replicon were cured without selection, while selection for DAP enhanced stability without detectable loss of any of the three resident virulence plasmids. The dapAX mutation rendered Y. pestis avirulent in mouse models of bubonic and septicemic plague which could be complemented when dapAX was inserted in single or multi-copy, restoring development of disease that was indistinguishable from the wild type parent strain. We further identified a high level, constitutive promoter in Y. pestis that could be used to drive expression of fluorescent reporters in dapAX strains that had minimal impact to virulence in mouse models while enabling sensitive detection of bacteria during infection. Thus, diaminopimelic acid selection for single or multi-copy genetic systems in Yersinia pestis offers an improved alternative to antibiotics for in vivo studies that causes minimal disruption to virulence

Modified Needle-Tip PcrV Proteins Reveal Distinct Phenotypes Relevant to the Control of Type III Secretion and Intoxication by Pseudomonas aeruginosa

The type III secretion system (T3SS) is employed to deliver effector proteins to the cytosol of eukaryotic hosts by multiple species of Gram-negative bacteria, including Pseudomonas aeruginosa. Translocation of effectors is dependent on the proteins encoded by the pcrGVHpopBD operon. These proteins form a T3S translocator complex, composed of a needle-tip complex (PcrV), translocons (PopB and PopD), and chaperones (PcrG and PcrH). PcrV mediates the folding and insertion of PopB/PopD in host plasmic membranes, where assembled translocons form a translocation channel. Assembly of this complex and delivery of effectors through this machinery is tightly controlled by PcrV, yet the multifunctional aspects of this molecule have not been defined. In addition, PcrV is a protective antigen for P. aeruginosa infection as is the ortholog, LcrV, for Yersinia. We constructed PcrV derivatives containing in-frame linker insertions and site-specific mutations. The expression of these derivatives was regulated by a T3S-specific promoter in a pcrV-null mutant of PA103. Nine derivatives disrupted the regulation of effector secretion and constitutively released an effector protein into growth medium. Three of these regulatory mutants, in which the linker was inserted in the N-terminal globular domain, were competent for the translocation of a cytotoxin, ExoU, into eukaryotic host cells. We also isolated strains expressing a delayed-toxicity phenotype, which secrete translocators slowly despite the normal level of effector secretion. Most of the cytotoxic translocation-competent strains retained the protective epitope of PcrV derivatives, and Mab166 was able to protect erythrocytes during infection with these strains. The use of defined PcrV derivatives possessing distinct phenotypes may lead to a better understanding of the functional aspects of T3 needle-tip proteins and the development of therapeutic agents or vaccines targeting T3SS-mediated intoxication

The Role of relA and spoT in Yersinia pestis KIM5+ Pathogenicity

The ppGpp molecule is part of a highly conserved regulatory system for mediating the growth response to various environmental conditions. This mechanism may represent a common strategy whereby pathogens such as Yersinia pestis, the causative agent of plague, regulate the virulence gene programs required for invasion, survival and persistence within host cells to match the capacity for growth. The products of the relA and spoT genes carry out ppGpp synthesis. To investigate the role of ppGpp on growth, protein synthesis, gene expression and virulence, we constructed a ΔrelA ΔspoT Y. pestis mutant. The mutant was no longer able to synthesize ppGpp in response to amino acid or carbon starvation, as expected. We also found that it exhibited several novel phenotypes, including a reduced growth rate and autoaggregation at 26°C. In addition, there was a reduction in the level of secretion of key virulence proteins and the mutant was>1,000-fold less virulent than its wild-type parent strain. Mice vaccinated subcutaneously (s.c.) with 2.5×104 CFU of the ΔrelA ΔspoT mutant developed high anti-Y. pestis serum IgG titers, were completely protected against s.c. challenge with 1.5×105 CFU of virulent Y. pestis and partially protected (60% survival) against pulmonary challenge with 2.0×104 CFU of virulent Y. pestis. Our results indicate that ppGpp represents an important virulence determinant in Y. pestis and the ΔrelA ΔspoT mutant strain is a promising vaccine candidate to provide protection against plague

Milwaukee Wholesale Fresh Fruit and Vegetable Market Facilities

Excerpts from the report: The wholesale fresh fruit and vegetable market of Milwaukee is divided into two areas: The Broadway Street market, located at Broadway and Detroit Streets, and Central Market, at Fifth and Vliet Streets. In Milwaukee, as in other market areas of the country, wholesale fresh fruit and vegetable facilities which many dealers occupy were neither designed nor intended to be used for wholesale food distribution. Many of the present facilities were built before modern motor and rail transportation was available. In the fall of 1963, this study was undertaken at the request of the city of Milwaukee and the Milwaukee Fresh Fruit and Vegetable Advertising Council, in cooperation with the Wisconsin State Department of Agriculture. This study has the following objectives: To analyze the present wholesale fresh fruit and vegetable operations in Milwaukee. To estimate the major costs of handling fresh fruits and vegetables under present conditions. To determine facilities and possible sites needed for efficient wholesale marketing of these products. To estimate the cost of constructing new facilities. To estimate potential savings or benefits which may accrue from any suggested improvements

Wholesale Food Distribution Facilities for Honolulu, Hawaii

Excerpts from the report Introduction: The wholesale food industry in Hawaii has long recognized the need for more modern facilities and improved handling practices in the State. Facilities that were designed for the needs of Hawaii as a territory cannot be considered adequate to serve the projected growth in population and increase of tourism. In 1964, a detailed study of food wholesaling in Honolulu was undertaken at the request of the State government. This study was under general supervision of the Transportation and Facilities Research Division, Agricultural Research Service, U.S. Department of Agriculture (USDA), in cooperation with the following State agencies: The Department of Land and Natural Resources, the Department of Economic Development, the Department of Agriculture, and the University of Hawaii, College of Tropical Agriculture. The purpose of the study was to determine (1) the adequacy of wholesale food facilities in Honolulu for present and future needs; (2) the costs of handling food products in existing facilities; (3) whether there is a need for new facilities; (4) how many wholesalers the center should provide for; (5) how much income would be needed to finance such a center; and (6) whether the Fort Armstrong site would be large enough; and (7) to outline the benefits that might accrue from improved facilities

Food Distribution Facilities for Oakland, California

Excerpts from the report: At the request of food wholesalers in Oakland, the Industrial Development Commission made inquiries of individual wholesale food firms in the city during the fall of 1966 to determine the extent of interest in building a new market that would house all types of food wholesalers. These inquiries showed substantial interest. As a result, Mayor John H. Reading requested the Transportation and Facilities Research Division, Agricultural Research Service, to make a complete study to determine what action should be taken to improve wholesale food distribution in the Oakland area. The study began in the spring of 1967. This study had the following objectives: 1. To determine the adequacy of food marketing facilities in the Oakland area for present and future needs. 2. To develop plans and designs for improved facilities. 3. To consider sites that would be adequate to provide the most efficient means of distributing food supplies to Oakland and the surrounding area. 4. To estimate construction costs, possible operating expenses, and rental requirements. 5. To estimate possible savings and other benefits from improved food-marketing facilities

Improved Food Distribution Facilities for Dallas, Texas

Excerpts from the report: This study was undertaken in the fall of 1972 at the request of the Dallas Fresh Fruit and Vegetable Association and industry representatives in cooperation with the Dallas Department of Planning and Urban Development and various economic and industrial development agencies in the city. Part of the present central produce district in Dallas is included in plans for urban improvement. If these plans are implemented, they will affect most fresh fruit and vegetable wholesalers now operating in the produce district. Although most fresh fruit and vegetable wholesalers are concentrated in a market area near downtown Dallas, all fresh fruit and vegetable wholesalers operating in the city were included in the study. Firms were not included if they were predominantly a retail operation. This study of facilities for handling fresh fruits and vegetables at wholesale in Dallas is part of a research program to improve the operations of food distributors and thus increase the efficiency of distributing fresh and processed fruits and vegetables to all consumers in the Dallas area. Data in this report were obtained from wholesale fresh fruit and vegetable firms through personal interview

New Bedford Wholesale Food-Distribution Facilities

Excerpts from the report: In New Bedford, the majority of the food handling facilities are located either in the path of Interstate Route 195 and the city connector, proposed route 140, both of which are programmed for early construction, or in an area presently under study for urban renewal and historic restoration. Most of the remaining firms are in areas for which renewal plans will be made in the future. In the fall of 1961, this study was undertaken at the request of the Mayor of New Bedford and in cooperation with the City Planning Department. The purpose of this report is to assist in planning wholesale food handling facilities to replace existing facilities which may be displaced by proposed highway construction, a historical restoration project, or future urban renewal project. This study is part of a broad program of marketing research to aid in reducing costs of marketing farm and food products. It has the following objectives: • To analyze the present food marketing situation and facilities in New Bedford. • To determine those facilities that would best suit present and future needs. • To estimate costs of facility construction, possible operating expenses, and self-liquidating potentials. • To estimate probable savings and other benefits from any suggested improvements

Wholesale Food Distribution Facilities for Montgomery, Ala.

Excerpts from the report: This study was undertaken in the spring of 1965 at the request of the city officials of Montgomery. It was conducted by the Agricultural Research Service of the U.S. Department of Agriculture and the Alabama State Department of Agriculture and Industries in cooperation with Auburn University. The report outlines plans for new and improved wholesale food handling facilities to replace existing unsuitable, outdated, or inefficient buildings that may be displaced by long-range plans for civic improvement. In Montgomery, more than 30 percent of the food volume of independent wholesalers is distributed from locations within the central business district, an area for which preliminary planning for urban renewal is underway. The study had the following objectives: • To analyze the present wholesale food marketing facilities in Montgomery. • To estimate the major costs of handling food products, under present conditions, by these wholesalers. • To determine facilities and possible sites needed for efficient wholesale marketing of these products. • To estimate the cost of constructing new facilities. • To estimate potential savings or benefits that may accrue from any suggested improvements