An immunoprotective molecule, the major secretory protein of Legionella pneumophila, is not a virulence factor in a guinea pig model of Legionnaires' disease.

We have examined whether a molecule that is capable of inducing immune protection, the major secretory protein (MSP) of Legionella pneumophila, is required for virulence in a guinea pig model of Legionnaires' disease. To do so, we have compared the virulence in guinea pigs of an isogenic pair of L. pneumophila, Philadelphia 1 strain, one of which produces MSP (MSP+) and one of which does not (MSP-). Both the MSP- strain and the MSP+ strain of L. pneumophila are highly virulent for guinea pigs, inducing similar signs and progression of illness. Both strains are lethal and have comparable LD50s and LD100s. Both strains multiply in the lungs of guinea pigs at a similar rate, and both strains produce indistinguishable pathological lesions in the lungs. Both strains maintain a stable phenotype with guinea pig passage, i.e., the MSP- strain does not regain the capacity to secrete MSP and the MSP+ strain retains its capacity to secrete MSP after lung passage. Although vaccination with MSP induces strong protective immunity in the guinea pig against lethal aerosol challenge with L. pneumophila, this protective immunogen is not required in its intact proteolytically active form for the expression of virulence by the intracellular pathogen L. pneumophila. This demonstrates that a protective immune response need not necessarily be directed against a virulence determinant and suggests that any molecule that allows the host immune system to detect and act against an intracellularly sequestered pathogen may potentially serve as a protective immunogen against such a pathogen.

How is the intracellular fate of the Legionella pneumophila phagosome determined?

The following pair of articles, the first by Gil Segal and Howard Shuman, and the second by James Kirby and Ralph Isberg (Trends Microbiol. 6, 256-258), explore the genetics and function of the icm/dot genes of Legionella pneumophila. This gene family is implicated in several aspects of virulence and appears to constitute components of a conjugal transfer system that has been adopted to prevent phagosome-lysosome fusion in the host cell and to mediate host cytotoxicity by pore formation. Whether these functions are natural consequences or operate in parallel remains to be discovered.

Sodium/proton antiport is required for growth of Escherichia coli at alkaline pH.

Evidence is presented indicating that Escherichia coli requires the Na+/H+ antiporter and external sodium (or lithium) ion to grow at high pH. Cells were grown in plastic tubes containing medium with a very low Na+ content (5-15 microM). Normal cells grew at pH 7 or 8 with or without added Na+, but at pH 8.5 external Na was required for growth. A mutant with low antiporter activity failed to grow at pH 8.5 with or without Na+. On the other hand, another mutant with elevated antiporter activity grew at a higher pH than normal (pH 9) in the presence of added Na+ or Li+. Amiloride, an inhibitor of the antiporter, prevented cells from growing at pH 8.5 (plus Na+), although it had no effect on growth in media of lower pH values.

Allele-specific malE mutations that restore interactions between maltose-binding protein and the inner-membrane components of the maltose transport system.

Active accumulation of maltose and maltodextrins by Escherichia coli depends on an outer-membrane protein. LamB, a periplasmic maltose-binding protein (MalE, MBP) and three inner-membrane proteins, MalF, MalG and MalK. MalF and MalG are integral transmembrane proteins, while MalK is associated with the inner aspect of the cytoplasmic membrane via an interaction with MalG. Previously we have shown that MBP is essential for movement of maltose across the inner membrane. We have taken advantage of malF and malG mutants in which MBP interacts improperly with the membrane proteins. We describe the properties of malE mutations in which a proper interaction between MBP and defective MalF and MalG proteins has been restored. We found that these malE suppressor mutations are able to restore transport activity in an allele-specific manner. That is, a given malE mutation restores transport activity to different extents in different malF and malG mutants. Since both malF and malG mutations could be suppressed by allele-specific malE suppressors, we propose that, in wild-type bacteria, MBP interacts with sites on both MalF and MalG during active transport. The locations of different malE suppressor mutations indicate specific regions on MBP that are important for interacting with MalF and MalG.

Genetic analysis of periplasmic binding protein dependent transport in Escherichia coli. Each lobe of maltose-binding protein interacts with a different subunit of the MalFGK2 membrane transport complex.

Escherichia coli is able to accumulate maltose and maltodextrins by an ATP-binding cassette transporter known as the maltose transport system. This transport system is comprised of five proteins: the LamB protein in the outer membrane; the periplasmic maltose-binding protein (MBP); two integral inner membrane proteins, MalF and MalG; and MalK, which is associated with the cytoplasmic face of the inner membrane. It has been previously suggested that MBP interacts with MalF and MalG during sugar transport across the inner membrane. In two independent genetic studies, reported here, residue 210 of MBP has been identified as an important site for its interaction with MalF. In one study, allele-specific suppressors of a malF mutation, malF506, were isolated and yielded mutations which altered residue tyrosine 210 of MBP to aspartic acid. In the other study, dominant mutations in malE (the structural gene of MBP) were isolated; one of these altered the same tyrosine residue (210) to cysteine. It was shown that the Y210C MBP mutant is also an allele-specific suppressor malF506, and that of the suppressor MBP alleles also exhibited dominant-negative phenotypes. Previously it was shown that alterations at residues glycine 13 and aspartate 14 of MBP can result in suppression of a malG mutant. From these results and those described, it is possible to propose a simple model in which the amino-terminal lobe of MBP interacts with MalG and the carboxy-terminal lobe of MBP interacts with MalF. The locations of residues 13, 14 and 210 on the three-dimensional structure of MBP are in keeping with this model.

Crystal structures and solution conformations of a dominant-negative mutant of Escherichia coli maltose-binding protein.

A mutant of the periplasmic maltose-binding protein (MBP) with altered transport properties was studied. A change of residue 230 from tryptophan to arginine results in dominant-negative MBP: expression of this protein against a wild-type background causes inhibition of maltose transport. As part of an investigation of the mechanism of such inhibition, we have solved crystal structures of both unliganded and liganded mutant protein. In the closed, liganded conformation, the side-chain of R230 projects into a region of the surface of MBP that has been identified as important for transport while in the open form, the same side-chain takes on a different, and less ordered, conformation. The crystallographic work is supplemented with a small-angle X-ray scattering study that provides evidence that the solution conformation of unliganded mutant is similar to that of wild-type MBP. It is concluded that dominant-negative inhibition of maltose transport must result from the formation of a non-productive complex between liganded-bound mutant MBP and wild-type MalFGK2. A general kinetic framework for transport by either wild-type MalFGK2 or MBP-independent MalFGK2 is used to understand the effects of dominant-negative MBP molecules on both of these systems.

The use of gene fusions of study bacterial transport proteins.

The transport of solutes by bacteria has been studied for about thirty years. Early experiments on amino acid entry and galactoside accumulation provided concrete evidence that bacteria possessed specific transport systems and that these were subject to regulation. Since than a large number of transport systems have been discovered and studied extensively. Many of these use entirely different strategies for capturing or accumulating substrates. This diversity reflects variation in the availability of nutrients an ions in the different environments tolerated and inhabited by microorganisms. Examination of a few bacterial transport systems provides an opportunity to gain insight into a wide range of topics in the area of membrane transport. These include: the identification of carrier proteins and their arrangement in the membrane, the regulation of transport protein synthesis by environmental factors, and the localization of transport proteins to their extracytoplasmic destinations. It has been possible to construct a number of bacterial strains in which the gene (lacZ) which codes for the cytoplasmic enzyme beta-galactosidase is fused to genes which code for transport proteins. The following article is intended to illustrate how these gene fusions have been used to study the regulation and structure of transport proteins in Escherichia coli.

Active transport of maltose in Escherichia coli K12. Role of the periplasmic maltose-binding protein and evidence for a substrate recognition site in the cytoplasmic membrane.

The active transport of maltose in Escherichia coli requires the products of five genes. These include a water-soluble periplasmic maltose-binding protein, three cytoplasmic membrane proteins, and an outer membrane protein. In order to evaluate the role of the maltose-binding protein in active transport, a nonpolar internal deletion of the structural gene for the maltose-binding protein was constructed. A strain which contains this deletion is unable to grow on maltose at an external concentration of 25 mM, even when the remaining components of the transport system are synthesized constitutively. This demonstrates that the periplasmic maltose-binding protein is essential for detectable translocation of maltose across the cytoplasmic membrane. Mal+ revertants of the deletion strain were obtained. In one of these strains, the remaining components of the maltose transport system gained the ability to translocate maltose across the membrane independently of the periplasmic binding protein. Maltose transport in this revertant strain is specific for maltose; it is not inhibited by other alpha and beta glucosides and galactosides. In contrast to the wild type, transport activity in the Mal+ revertant strain is retained by spheroplasts. The cytoplasmic membrane components of the maltose transport system in the revertant appear to form a substrate recognition site. It is likely that this site exists in wild type cells but is available only to substrate molecules that are bound to the maltose-binding protein. A model for the operation of the transport system is presented. In this model, the substrate recognition site in the cytoplasmic membrane is exposed to alternate sides of the membrane.

The maltose-maltodextrin transport system of Escherichia coli.

The general features of the active transport system for maltose and maltodextrins are described. Two cytoplasmic membrane components have been identified with the aid of lacZ-gene fusions. One protein (MalF) is an integral membrane protein. The other protein (MalK) seems to be a peripheral membrane protein on the inner aspect of the membrane.

Overproduction of MalK protein prevents expression of the Escherichia coli mal regulon.

The mal regulon of Escherichia coli comprises a large family of genes whose function is the metabolism of linear maltooligosaccharides. Five gene products are required for the active accumulation of maltodextrins as large as maltoheptaose. Two cytoplasmic gene products are necessary and sufficient for the intracellular catabolism of these sugars. Two newly discovered enzymes have the capacity to metabolize these sugars but are not essential for their catabolism in wild-type cells. A single regulatory protein, MalT, positively regulates the expression of all of these genes in response to intracellular inducers, one of which has been identified as maltotriose. In the course of studying the mechanism of the transport system, we have placed the structural gene for one of the transport proteins, MalK, under the control of the Ptrc promoter to produce large amounts of this protein. We found that although high-level expression of MalK was not detrimental to E. coli, the increased amount of MalK decreased the basal-level expression of the mal regulon and prevented induction of the mal system even in the presence of external maltooligosaccharides. Constitutive mutants in which MalT does not depend on the presence of the internal inducer(s) were unaffected by the increased levels of the MalK protein. These results are consistent with the idea that MalK protein somehow interferes with the activity of the MalT protein. Different models for the regulatory function of MalK are discussed.

Isolation of a Legionella pneumophila restriction mutant with increased ability to act as a recipient in heterospecific matings.

The ability of Legionella pneumophila to act as a recipient of IncP and IncQ plasmids in matings with Escherichia coli varies widely from strain to strain. We found that the low efficiency of mating of the Philadelphia-1 strain is due to a type II restriction-modification system, and we isolated and characterized a Philadelphia-1 mutant that lacks the restriction enzyme activity.

Unliganded maltose-binding protein triggers lactose transport in an Escherichia coli mutant with an alteration in the maltose transport system.

Escherichia coli accumulates malto-oligosaccharides by the maltose transport system, which is a member of the ATP-binding-cassette (ABC) superfamily of transport systems. The proteins of this system are LamB in the outer membrane, maltose-binding protein (MBP) in the periplasm, and the proteins of the inner membrane complex (MalFGK2), composed of one MalF, one MalG, and two MalK subunits. Substrate specificity is determined primarily by the periplasmic component, MBP. However, several studies of the maltose transport system as well as other members of the ABC transporter superfamily have suggested that the integral inner membrane components MalF and MalG may play an important role in determining the specificity of the system. We show here that residue L334 in the fifth transmembrane helix of MalF plays an important role in determining the substrate specificity of the system. A leucine-to-tryptophan alteration at this position (L334W) results in the ability to transport lactose in a saturable manner. This mutant requires functional MalK-ATPase activity and the presence of MBP, even though MBP is incapable of binding lactose. The requirement for MBP confirms that unliganded MBP interacts with the inner membrane MalFGK2 complex and that MBP plays a crucial role in triggering the transport process.

Characterization of a new region required for macrophage killing by Legionella pneumophila.

In a previous study, a collection of 55 Legionella pneumophila mutants defective for macrophage killing was isolated by transposon mutagenesis. In this study, nine of these mutants that belong to the same DNA hybridization group (group 3) were characterized. A wild-type DNA fragment that covers this DNA hybridization group was cloned and sequenced. This region was found to contain six new genes (designated icmT, icmS, icmR, icmQ, icmP, and icmO), five of which contain at least one transposon insertion. No transposon insertion was found in icmS. However, this gene was found to be required for macrophage killing, since a kanamycin resistance cassette introduced into icmS by gene replacement resulted in a mutant that was attenuated for macrophage killing. A plasmid containing the DNA fragment that covers this region complements all the mutants for macrophage killing, although various levels of complementation were observed for mutants in different genes. Complementation tests were also performed with plasmids containing one or two of these genes, as well as with plasmids containing nonpolar in-frame deletions. The results from these complementation tests indicated that all six genes located in this region are needed for macrophage killing and that they are probably arranged as two transcriptional units (icmTS and icmPO) and two genes (icmR and icmQ). A region upstream of the coding sequence of several icm genes may contain a potential promoter and/or regulatory site. Homology searches show that icmP and icmO bear significant homology to the trbA and trbC genes from the Salmonella R64 plasmid, respectively. The sequences of the other four genes do not show significant homology with any entries in sequence databases.

Truncation of MalF results in lactose transport via the maltose transport system of Escherichia coli.

The active accumulation of maltose and maltodextrins by Escherichia coli is dependent on the maltose transport system. Several lines of evidence suggest that the substrate specificity of the system is not only determined by the periplasmic maltose-binding protein but that a further level of substrate specificity is contributed by the inner membrane integral membrane components of the system, MalF and MalG. We have isolated and characterized an altered substrate specificity mutant that transports lactose. The mutation responsible for the altered substrate specificity results in an amber stop codon at position 99 of MalF. The mutant requires functional MalK-ATPase activity and hydrolyzes ATP constitutively. It also requires MalG. The data suggest that in this mutant the MalG protein is capable of forming a low affinity transport path for substrate.

The Legionella pneumophila major secretory protein, a protease, is not required for intracellular growth or cell killing.

The Legionella pneumophila major secretory protein (Msp) is a Zn2+ metalloprotease whose function in pathogenesis is unknown. The structural gene for the Msp protease, mspA, was isolated from an L. pneumophila genomic library. In Escherichia coli which contain plasmids with the mspA gene, Msp protein and activity are found in the periplasmic space and the cytoplasm. Transposon mutagenesis with Tn9 of an mspA-containing plasmid in E. coli yielded mutants which no longer expressed protease activity and others with increased protease activity. These results suggested that mspA expression might be regulated. Msp was shown to be produced at a much higher level in L. pneumophila grown in rich compared to semidefined media. A Tn9 insertion which abolishes Msp expression was introduced into the L. pneumophila genome. This mspA::Tn9 L. pneumophila strain showed no detectable production of Msp by immunoblot analysis, and it had less than 0.1% of the protease activity found in the wild-type strain. This mutant was fully capable of growing within and killing human macrophages derived from the HL-60 cell line.