Puromycin-N-acetyltransferase as a selectable marker for use in Plasmodium falciparum.

The limited number of selectable markers available for malaria transfection has hindered extensive manipulation of the Plasmodium falciparum genome and subsequently thorough genetic analysis of this organism. In this paper, we demonstrate that P. falciparum is highly sensitive to the drug puromycin, but that transgenic expression of the puromycin-N-acetyltransferase (PAC) gene from Streptomyces alboninger confers resistance to this drug with the IC(50) and IC(90) values increasing approximately 3- and 7-fold, respectively in PAC-expressing parasites. Despite this relatively low level of resistance, parasite populations transfected with the PAC selectable marker and selected directly on puromycin emerged at the same rate post-transfection as human dihydrofolate reductase (hDHFR)-expressing parasites, selected independently with the anti-folate drug WR99210. Transfected parasites generally maintained the PAC expression plasmid episomally at between two and six copies per parasite. We also demonstrate by cycling transfected parasites in the presence and absence of puromycin for several weeks, that the PAC selectable marker can be used for gene-targeting. Since the mode of action of puromycin is distinct from other drugs currently used for the stable transfection of P. falciparum, the PAC selectable marker should also have applicability for use in conjunction with other positive selectable markers, thereby increasing the possibilities for more complex functional studies of this organism.

P25 and P28 proteins of the malaria ookinete surface have multiple and partially redundant functions.

The ookinete surface proteins (P25 and P28) are proven antimalarial transmission-blocking vaccine targets, yet their biological functions are unknown. By using single (Sko) and double gene knock-out (Dko) Plasmodium berghei parasites, we show that P25 and P28 share multiple functions during ookinete/oocyst development. In the midgut of mosquitoes, the formation of ookinetes lacking both proteins (Dko parasites) is significantly inhibited due to decreased protection against lethal factors, including protease attack. In addition, Dko ookinetes have a much reduced capacity to traverse the midgut epithelium and to transform into the oocyst stage. P25 and P28 are partially redundant in these functions, since the efficiency of ookinete/oocyst development is only mildly compromised in parasites lacking either P25 or P28 (Sko parasites) compared with that of Dko parasites. The fact that Sko parasites are efficiently transmitted by the mosquito is a compelling reason for including both target antigens in transmission-blocking vaccines.

Antibodies against merozoite surface protein (MSP)-1(19) are a major component of the invasion-inhibitory response in individuals immune to malaria.

Antibodies that bind to antigens expressed on the merozoite form of the malaria parasite can inhibit parasite growth by preventing merozoite invasion of red blood cells. Inhibitory antibodies are found in the sera of malaria-immune individuals, however, the specificity of those that are important to this process is not known. In this paper, we have used allelic replacement to construct a Plasmodium falciparum parasite line that expresses the complete COOH-terminal fragment of merozoite surface protein (MSP)-1(19) from the divergent rodent malaria P. chabaudi. By comparing this transfected line with parental parasites that differ only in MSP-1(19), we show that antibodies specific for this domain are a major component of the inhibitory response in P. falciparum-immune humans and P. chabaudi-immune mice. In some individual human sera, MSP-1(19) antibodies dominated the inhibitory activity. The finding that antibodies to a small region of a single protein play a major role in this process has important implications for malaria immunity and is strongly supportive of further understanding and development of MSP-1(19)-based vaccines.

Complementation of Plasmodium berghei TRAP knockout parasites using human dihydrofolate reductase gene as a selectable marker.

Previously we have used the Plasmodium dihydrofolate reductase thymidylate synthase (DHFR-TS) selectable marker to generate Plasmodium berghei TRAP null mutant parasites. These TRAP null mutants do not glide and they showed a great reduction in their ability to infect mosquito salivary glands and the hepatocytes of the vertebrate host. Thus far, complementation of these knockout parasites was not possible due to the lack of additional selectable markers. Recently, a new selectable marker, based on the human dihydrofolate reductase (hDHFR) gene, has been developed which confers resistance to the antifolate drug WR99210. This drug has been found to be highly active against pyrimethamine-sensitive and -resistant strains of P. berghei. In this study, we have used the hDHFR gene as a second selectable marker for the complementation of P. berghei TRAP null mutant parasites. Restoration of the TRAP null mutant parasites to the wild-type phenotype was achieved in this study via autonomously replicating episomes bearing a wild-type copy of the TRAP gene. This is the first report of complementation of a mutant phenotype in malaria parasites.

The development of genetic tools for dissecting the biology of malaria parasites.

Plasmodium parasites are haploid unicellular organisms that cause malaria. In the last decade, transfection systems have been developed for both human and animal model species of Plasmodium, providing a broad range of genetic tools for the study of malaria parasite biology. Transient transfection has been used to provide insight into the regulation of gene expression by Plasmodium spp. The development of stable transfection technologies has provided the opportunity to express transgenes in Plasmodium spp., as well as elucidate the function of proteins by disrupting, modifying, or replacing the genes encoding them. These genetic tools represent an important breakthrough for malaria research and will significantly contribute to our understanding of the biology of the parasite. However, further developments in this technology are still required, especially because the full genome sequence of the major human malaria parasite Plasmodium falciparum will shortly be available. Ultimately, the biological information obtained through genetic manipulation of Plasmodium spp. will facilitate a more rational approach to vaccine and drug design.

The selectable marker human dihydrofolate reductase enables sequential genetic manipulation of the Plasmodium berghei genome.

Genetic transformation of malaria parasites has been limited by the number of selectable markers available. For the rodent malaria parasite, Plasmodium berghei, only a single selection marker has been at hand, utilising the dihydrofolate reductase-thymidylate synthase gene from either P. berghei or Toxoplasma gondii to confer resistance to the anti-malarial drug pyrimethamine. Here we report the use of the human dihydrofolate reductase (hDHFR) gene as a new selectable marker, which confers resistance to the antifolate inhibitor WR99210 upon both pyrimethamine sensitive and resistant isolates of P. berghei. Transfection with circular constructs containing the hDHFR gene resulted in the generation of highly resistant parasites containing multiple copies of episomally-maintained plasmids. These parasites showed around a 1000-fold increase in resistance to WR99210 compared to the parental parasites. We were also able to generate and select transgenic parasites harbouring only a single copy of hDHFR targeted into their genome, despite the fact that these parasites showed only a fivefold increase in resistance to WR99210 compared to the parental parasites. Importantly, and for the first time with malaria parasites, the hDHFR gene could be used in conjunction with the existing pyrimethamine selectable markers. This was demonstrated by reintroducing the circumsporozoite (CS) gene into transgenic CS-knockout mutant parasites that contained the P. berghei DHFR-TS selectable marker. The development of hDHFR as a second selectable marker will greatly expand the use of transformation technology in Plasmodium, enabling more extensive genetic manipulation and thus facilitating more comprehensive studies on the biology of the malaria parasite.

Effect of bacterial invasion of macrophages on the outcome of assays to assess bacterium-macrophage interactions.

In vitro assays to quantify killing of bacteria by macrophages provide useful insights into host-pathogen relations. In the present study, we used strains of Yersinia enterocolitica and Escherichia coli which varied in their ability to invade mammalian cells to evaluate these assays. The results showed that 30 min and 24 h after incubation with murine bone marrow-derived macrophages, strains of Y. enterocolitica and E. coli which expressed invasin (an outer membrane protein which allows bacteria to penetrate mammalian cells) achieved significantly greater numbers in macrophages than otherwise isogenic bacteria which lacked this protein (P < 0.01). When the 24-h data were corrected for the number of bacteria ingested by macrophages initially, the differences between invasin-positive and -negative bacteria were no longer evident (P> 0.2). This study has shown (1) that invasin-mediated penetration of macrophages by bacteria is not associated with enhanced intracellular survival, and (2) that invasion of macrophages by bacteria may influence the interpretation of assays for bactericidal capacity unless allowance is made for the number of bacteria ingested during the early phase of the assay.

Hemolytic-uremic syndrome following urinary tract infection with enterohemorrhagic Escherichia coli: case report and review.

A 6-week-old child with acute urinary tract infection caused by Shiga toxin-producing Escherichia coli (STEC) O5:H-developed hemolytic-uremic syndrome (HUS). Molecular and phenotypic analysis of the urinary isolate indicated that it lacked uropathic properties and that it was probably of intestinal origin. Nevertheless, the patient did not experience a diarrheal prodrome, nor was STEC or Shiga toxin detected in his feces at any time. Examination of the patient's serum pointed to recent infection with E. coli O5, with no evidence of exposure to E. coli O157, O111, or O26. A review of 13 previously reported cases of HUS associated with acute urinary tract infection indicated that this was the first case of nondiarrheal HUS in which infection with the most common STEC serogroups was specifically excluded. This case illustrates the need to investigate patients with nondiarrheal HUS for infection with STEC.

A novel mechanism of urease regulation in Yersinia enterocolitica.

Yersinia enterocolitica produces the enzyme urease which hydrolyses urea, resulting in the production of carbonic acid and ammonia and a net increase in pH. In the presence of urea, urease enhances survival of Y. enterocolitica in the stomach and presumably in other acidic environments the bacteria encounter during the course of infection. In this study we show that Y. enterocolitica urease is a cytosolic enzyme which has a low Km value (0.15 +/- 0.01 mM urea), suggesting that it functions at close to maximum velocity even at the low concentrations of urea available to Y. enterocolitica in gastric fluid and other tissues. Y. enterocolitica urease was active over a wide pH range, but unlike most other bacterial ureases, displayed an optimal activity at pH 3.5-4.5, suggesting a physiological role in protecting the bacteria from acid. Higher levels of urease activity were attained at 28 degrees C than at 37 degrees C, and investigation of the regulation of urease production revealed that the enzyme was not induced by urea, or by nitrogen limitation. Instead maximal activity was attained during the stationary phase of growth which coincides with the period of maximum acid tolerance of the bacteria. This type of regulation has not been described for any other ureolytic bacteria and seems to be unique to Y. enterocolitica.

Analysis of the urease gene complex of members of the genus Yersinia.

The urease gene complex of Yersinia enterocolitica is relatively conserved within the species, although this conservation may not extend to other members of the genus. Spontaneous urease-negative isolates of Y. enterocolitica appear to have arisen as a result of large deletions within this complex, while Y. pestis shows no significant deletions within the complex, despite being urease negative.

Contribution of urease to acid tolerance in Yersinia enterocolitica.

The stomach serves as a barrier to enteric infection because of the antibacterial effect of the hydrochloric acid in gastric juice. In this study, we tested the ability of the enteric pathogen Yersinia enterocolitica to tolerate a pH range of 2.0 to 6.0 and found that under the conditions of a normal human fasting stomach (pH < 3 and a gastric emptying time of 2 h), Y. enterocolitica is highly acid resistant, showing approximately 85% survival. The resistance of Y. enterocolitica to acid in vitro depended on the bacterial growth phase and the concentration of urea in the medium, being maximal during stationary phase in the presence of at least 0.3 mM urea. Urease-negative mutants of Y. enterocolitica were constructed by disrupting the urease gene complex of a virulent strain of serogroup O9. Compared with the wild type, these mutants showed an approximately 1,000-fold decrease in the ability to tolerate acid in vitro (< 0.08% survival) and a 10-fold reduction in viability after passage through the stomachs of mice. Complementation of the disrupted urease genes in trans restored the ability of urease-negative mutants to tolerate low pH in vitro and gastric acidity to approximately wild-type levels. These findings indicate that urease is responsible for acid resistance in Y. enterocolitica and suggest that urease contributes to the virulence of Y. enterocolitica by enhancing the likelihood of bacterial survival during passage through the stomach.

Changes in the hemagglutinin gene of the neurovirulent influenza virus strain A/NWS/33.

The neurovirulent strain of influenza A virus, A/NWS/33, is able to infect a large range of cell types, including mouse brain cells, which are not infected by its parent, A/WS/33. This seems to be largely due to the hemagglutinin of A/NWS/33. The complete nucleotide sequence of the HA genes of both strains has been determined and a comparison revealed a number of changes. Analysis showed that the virulence capabilities of the NWS HA involve at least three different mechanisms: (a) loss of a glycosylation site; (b) a change at the cleavage site; and (c) a substitution in HA2, which may increase the pH of fusion.

Characterisation of the urease-encoding gene complex of Yersinia enterocolitica.

A cosmid gene library of chromosomal DNA from Yersinia enterocolitica A2635 (serogroup O:8) was constructed in Escherichia coli. Subcloning of a urease-positive (Ure+) clone revealed a region of 6.6 kb that was sufficient for expression of Ure activity in E. coli. Sequencing of this fragment disclosed seven ORFs transcribed in the same direction. On the basis of homology to known Ure, these were designated ureA, ureB, ureC, ureE, ureF, ureG and ureD, which are predicted to encode polypeptides of 11.1, 17.9, 61.0, 29.5, 25.0, 24.1 and 36.4 kDa, respectively. The polypeptides encoded by the ure gene complex of Y. enterocolitica are significantly divergent from those encoded by the ure operons of other Enterobacteriaceae, which appear to be closely related to each other. This suggests that the ure genes were acquired by Y. enterocolitica from an unrelated organism or alternatively, that they diverged from those of other Enterobacteriaceae some considerable time ago.