Invasiveness of Salmonella typhi strains in HeLa S3 monolayer cells

The internalization and intracellular multiplication, i.e., the invasiveness, of Salmonella typhi strains recently isolated from typhoid fever patients were confirmed in HeLa cell monolayers. When stained with Giemsa solution, intracellular bacteria were 0.6 X 1.2 micron in size and stained purple, whereas extracellular bacteria associated or not with the HeLa cell surface were 1.0 X 3.0 micron and stained deep blue. Strain GIFU 10007 was internalized into 23% of the HeLa cells within 10 min after inoculation. About 90% of the HeLa cells were infected after 24 hr incubation in kanamycin (KM)-containing medium. Intracellular multiplication of the challenge organism was verified by a large number of intracellular bacteria after 24 hr incubation in KM-containing medium by both light-microscopy of the Giemsa stained preparation and viable counts of intracellular bacteria. The viable counts of strain 10007 showed an increase of more than 40-fold within 24 hr after inoculation, whereas in the four other less or non-infective strains, recovery of viable bacteria was poor or nil. Strains which were highly invasive usually failed to show strong adhesion. The contribution of Vi antigen to the internalization of challenge organisms was not proved. Infective strains, when killed by formalin were still adhesive, but were not internalized. The same strains, when killed by boiling, were neither adhesive nor internalized. From these findings it was concluded that the internalization and multiplication of infective S. typhi strains in cultured HeLa cells should be regarded as an invasion rather than phagocytosis by host cells, and such invasiveness could be an indicator to estimate the virulence of S. typhi strains.     

Cytopathogenic effect of Salmonella typhi GIFU 10007 on M cells of murine ileal Peyer's patches in ligated ileal loops: an ultrastructural study

An electron microscopic study revealed that, within 30 min after inoculation into the ligated ileal loop of anesthetized mice, cells of Salmonella typhi GIFU 10007 adhered to the M cell surface of Peyer's patch lymphoid follicle epithelium, and induced almost complete destruction of M cells. The M cell cytoplasms were pinched off and extruded from the epithelial lining into the luminal space together with the lymphoid cells primarily enfolded into the corresponding M cells. When two or more M cells were destroyed, a large defect in the epithelial lining was apparent, and a number of bacteria appeared near the basal lamina of the epithelial lining. These findings suggest, as far as anesthetized murine ileal loops and strain 10007 are concerned, that ileal M cells are the target cell at an early stage of S. typhi infection and the infection may further progress to deeper tissues and to the general circulation.     

Vi-Suppressed wild strain Salmonella typhi cultured in high osmolarity is hyperinvasive toward epithelial cells and destructive of Peyer's patches

Salmonella typhi GIFU10007-3 which lost a viaB locus on its chromosome became highly invasive in our previous study. To investigate the phenomenon, we controlled Vi expression in wild strain S. typhi GIFU10007, and studied the invasive phenotype both in vitro and in vivo. When the wild strain of S. typhi was cultured in 300 mM NaCl containing Luria-Bertani broth (LBH), the expression of Vi antigen was suppressed, but secretion of invasion proteins (SipC, SipB and SipA) was increased. In this condition, wild strain S. typhi became highly invasive toward both epithelial cells and M cells of rat Peyer's patches. When GIFU10007 was cultured under conditions of high osmolarity, the bacteria disrupted Peyer's patches and induced massive bleeding in these structures only 20 min after inoculation into the ileal loop. In contrast, Vi-encapsulated wild strain GIFU10007 cultured under low osmolarity was not destructive, even after 60 min. To understand the role of the type III secretion system under conditions of high osmolarity, we knocked out the invA and sipC genes of both GIFU10007 and GIFU10007-3. Neither invA nor sipC mutants could invade epithelial cells or M cells in a high osmolarity environment. Our data show that the highly invasive phenotype was only expressed when the wild strain S. typhi was cultured under high osmolarity, which induced a state of Vi suppression, and in the presence of the type III secretion system.     

Lethality in mammalian cells due to hyperthermia under oxic and hypoxic conditions.

From several studies of hyperthermia there have been reports that hypoxic cells are more sensitive to heat than their oxic counterparts. Experimental techniques in this investigation eliminate the effect of pH, trypsinization and cell attachment, when assaying the effects of hyperthermia on cells. Under hypoxic conditions, HeLa S3 and Chinese hamster cell-lines do not have an increased sensitivity to heat compared with oxic cells. HeLa S3 cells are protected against heat by hypoxia. Light-microscopy indicates the rupture of the plasma membrane, occasional nuclear budding, membrane vesicles and granulation of cell contents after heating at 43 degrees C for 3 hours. Scanning electron micrographs show that cells are more rounded after heat treatment and that there is an accompanying decrease in the number of microvilli, suggesting that the mechanism of cell attachment is affected. Heated cells should be delicately handled and subjected to the minimal trauma so that an accurate comparison of survival can be made.     

Apoptosis of human keratinocytes after bacterial invasion

In this study, we examined the invasive capacity of Staphylococcus aureus and Salmonella typhi in human keratinocytes and monitored the number of viable intracellular bacteria at different post-infection times. The strains tested entered keratinocytes; both S. typhi and S. aureus were internalized within 30 min to 2 h after infection. No intracellular multiplication was observed, but S. typhi and S. aureus remained viable 72 h after infection. We also demonstrated that keratinocyte death following S. typhi and S. aureus invasion occurs by apoptosis as shown by DNA fragmentation. After 24 h of infection with S. typhi, the number of cells undergoing apoptosis were higher compared to infection with S. aureus. For prolonged infection times (48 h, 72 h) with both bacteria, there was no significant change in the number of cells undergoing apoptosis. The results demonstrated that viable intracellular S. typhi and S. aureus induced apoptosis in keratinocyte cells.     

Green fluorescent protein-based direct viable count to verify a viable but non-culturable state of Salmonella typhi in environmental samples.

The gfp-tagging method and lux-tagging method were compared to select a better method for verifying a viable but nonculturable (VBNC) state of bacteria in the environment. An environmental isolate of Salmonella typhi was chromosomally marked with a gfp gene encoding green fluorescent protein (GFP). The hybrid transposon mini-Tn5 gfp was transconjugated from E. coli to S. typhi. Using the same method, S. typhi was chromosomally marked with luxAB genes encoding luciferase. The survival of gfp-tagged S. typhi introduced into groundwater microcosms was examined by GFP-based plate count, total cell count, and a direct viable count method. In microcosms containing lux-tagged S. typhi, luminescence-based plate count and the measurement of bioluminescence of each microcosm sample were performed. In microcosms containing lux-tagged S. typhi, viable but nonculturable cells could not be detected by using luminometry. As no distinguishable luminescence signals from the background signals were found in samples containing no culturable cells, a VBNC state of S. typhi could not be verified in lux-based systems. However, comparison between GFP-based direct viable counts and plate counts was a good method for verifying the VBNC state of S. typhi. Because GFP-based direct viable count method provided a direct and precise estimation of viable cells of introduced bacteria into natural environments, it can be used for verifying the VBNC state of bacteria in environmental samples.     

Adherence to HEp-2 cells and replication in macrophages of Salmonella derby of human origin.

Adherence to a HEp-2 cell monolayer was tested for in four strains of Salmonella derby which were isolated from patients with diarrhea. One strain, SB1, was highly adherent and another strain, SB4, was nonadherent. The other two strains exhibited moderate adherence. Further in vitro study of invasion of HEp-2 cells by S. derby and its replication in murine peritoneal macrophages was carried out using SB1 and SB4. Thin section electron micrographs revealed that SB1 invaded HEp-2 cells but SB4 did not. The number of viable bacteria within macrophages was determined at intervals after inoculation of bacteria. The result indicates that SB1 can replicate in the macrophages but SB4 cannot. Flagella and fimbriae were compared by electron microscopy between SB1 and SB4, and their lipopolysaccharides and outer membrane proteins were also compared with each other by SDS-polyacrylamide gel electrophoresis. The presence of a 41 kDa protein in the outer membranes of SB1 was only the difference detected, suggesting that this protein could be a factor required for adherence of this serovar to epithelial cells.     

Biological and morphological aspects of the growth of equine abortion virus

Darlington, R. W. (St. Jude Children's Research Hospital, Memphis, Tenn.), and C. James. Biological and morphological aspects of the growth of equine abortion virus. J. Bacteriol. 92:250-257. 1966.-The growth of equine abortion virus (EAV) was studied by bioassay and electron microscopy in L-cell monolayer and suspension cultures, and in HeLa and BHK 21/13 cell monolayers. Results of virus assay (plaque-forming units) indicated that production of cell-associated virus (CAV) began at 6 to 9 hr after infection in all of the cell strains used. Virus release occurred 1 to 2 hr later. By 15 to 20 hr after infection, the amount of released virus (RV) equaled or surpassed that of CAV in all cells other than the HeLa cells, where the amount of RV did not equal CAV until 48 hr after infection. Electron microscopy of infected cells revealed no differences in the morphology of virus development in any of the cells used. Developing virus particles were first detected in cell nuclei at 9 hr after infection. At 12 hr, virus particles could be seen budding from the inner nuclear envelope. Budding into cytoplasmic vacuoles was not seen. Budding virus, virus in cytoplasmic vacuoles, and extracellular virus were all approximately 145 mmu in diameter, and were indistinguishable morphologically. These results indicated that EAV is quite similar to herpes simplex virus with respect to growth and morphology, and that the inner nuclear membrane is the principal site of virus envelopment.     

Pathogenic effect of enterotoxigenic Escherichia coli and Escherichia coli causing infantile diarrhoea.

Macroscopic, light and electron microscopic alterations in ligated rabbit intestinal loops challenged with five standard enterotoxigenic Escherichia coli (ETEC) and twenty-three enteropathogenic E. coli (EEC-I) strains, freshly isolated from infantile enteritis cases, were investigated. Only two O26 : K60 : H11 strains produced enterotoxin. Their living cultures, sterile filtrates of the fluid medium and ultrasonic lysates of the bacteria resulted in pronounced hypersecretion of the intestinal epithelium followed by fluid accumulation and loop dilatation. These two E. coli strains, similarly as the other loop-negative EEC-I strains, were able to penetrate into the intestinal epithelium. In contrast to the standard ETEC strains, the EEC-I bacteria, adhering to the brush border, intruded into the microvilli, multiplied on the outer epithelial cell membrane making close contact with it and, causing, shedding of microvilli, penetrated into enterocytes becoming enclosed in membrane-bound phagosome-like vacuoles, appeared in the lamina propria and elicited mild focal polymorphonuclear infiltration.     

Ultrastructure of soybean nodules. I: release of rhizobia from the infection thread

Root nodules on soybeans (var. Clark 63) were examined by transmission electron microscopy 10-12 days after seed inoculation and planting. The cell infection process appeared identical in both effective nodules, induced by Rhizobium japonicum strain 138 (USDA) and in ineffective nodules, induced by strain 8-0 (Iowa). Electron micrographs are presented which suggest that rhizobia are freed from the infection thread by disintegration of the thread wall and compartmentalization of the distintegrated wall material in membrane-bound vesicles derived from the membrane surrounding the thread. As the thread wall is removed in this manner, the bacteria are released into the host cytoplasm by a process which encloses each in an envelope also dervide from the thread membrane. Any thread wall material remaining around a bacterium after it has dissociated from the thread is removed from the envelope space by vesiculation of the membrane envelope. thus, it appears that endocytosis of both the bacteria and the material composing the infection thread wall occurs during release of rhizobia into the host cell.     

Ultrastructural aspects of the process of Salmonella typhi bacterial interaction with L929-line cells

The study of the use of scanning electron microscopy and the analysis of the initial stages of interaction between S. typhi and eukaryotic cells by the method of three-dimensional reconstruction has revealed that the infective agent penetrates into the cytoplasm on the principle of internalization. The internalization of S. typhi occurs with the active participation of the eukaryotic cells which, at the beginning, envelopes the bacteria with its processes, and the infective agents firmly adhere to the glycocalyx of the host cell by means of special fimbria-like formations differing from fimbriae by their lesser rigidity and thickness; then the microbes fixed to the membrane penetrate inside the cell without destroying its cytoplasmic membrane. Differences in the processes of the interaction of eukaryotic cells with S. typhi initial strain 238 and its variant free from the plasmid with a molecular weight of 6 Md, characterized by its lower capacity for association with cells of continuous cell culture L929, have been revealed. The factors stimulating the ingestion of S. typhi by eukaryotic cells are under study at present.     

The ability of some Yersinia enterocolitica strains to invade HeLa cells

Many types of Yersinia enterocolitica have been isolated from animal, environmental, food, and human sources but their public health significance remains uncertain. Seventy two strains of Y. enterocolitica were tested for their abilities to invade HeLa cells. The typical clinical strains invade HeLa cells like the other species of invasive pathogens. This characteristic remains even in old stock cultures and can be temperature-sensitive like the motility characteristic. With the use of electron micrographs it was demonstrated that the bacteria were truly intracellular and not merely adhering to the HeLa cell membrane. The esculin-and salicin-positive typical clinical strains did not invade HeLa cells. None of 34 food and water isolates were invasive by this test. The negative Y. enterocolitica strains did not adhere to the cells and cause ambiguous results. The HeLa cell test is simple, inexpensive, rapid, and should prove useful marker for screening the Y. enterocolitica isolates.     

Identification of challenged subarachnoid free cells.

Three distinct types of free cell contours are recognizable in scanning electron microscopy (SEM) on the leptomeningeal sheaths of dogs twelve days after an intrathecal injection of bacillus of dogs twelve days after an intrathecal injection of bacillus Calmette-Guerin (BCG). Macrophages posses abundant plasmalemnal blebs which are shown in transmission electron microscopy (TEM) to be composed of large membrane-bound vacuoles. Smooth surfaced lymphoblasts exhibit many basal microvilli that rest upon and often indent the plasmalemma of an underlying pial cell. Neutrophils display many microvilli over their rounded, chrysanthemum-like surfaces. The consistency with which these external features are expressed suggests that each cell type possesses characteristic surface topography, at least under these conditions of challenge.     

High-frequency intracellular invasion of epithelial cells by serotype M1 group A streptococci: M1 protein-mediated invasion and cytoskeletal rearrangements

A clonal variant of serotype M1 group A streptococcus (designated M1inv+) has been linked to severe and invasive infections, including sepsis, necrotizing fasciitis and toxic shock. High frequency internalization of cultured epithelial cells by the M1inv+ strain 90-226 is dependent upon the M1 protein. Invasion of HeLa cells was blocked by an anti-M1 antibody, invasion by an M1- strain (90-226 emm1::km) was greatly reduced, and latex beads bound to M1 protein were readily internalized by HeLa cells. Beads coated with a truncated M1 protein were internalized far less frequently. Scanning electron microscopy indicated that streptococci invade by a zipper-like mechanism, that may be mediated by interactions with host cell microvilli. Initially, internalized streptococci and streptococci undergoing endocytosis are associated with polymerized actin. Later in the internalization process, streptococcal-containing vacuoles are associated with the lysosomal membrane glycoprotein, LAMP-1.     

Function of coated membranes and multivesicular bodies during membrane regulation in stimulated exocrine pancreas cells

Summary Stimulation of secretion by pilocarpine results in a 70% loss of zymogen granules from pancreatic acinar cell during the first hr after injection of the drug. In previous work (Geuze and Poort, 1973), we found that the amount of membrane stored in the surface of the microvilli and of the numerous infoldings present in highly stimulated cells, increases during the first 2 hr and then decreases again during the 3rd hr after stimulation, concurrently with maximal endocytosis of sorbitol-[su14C].Further observations on the fine structure of stimulated cells at various time intervals after injection of pilocarpine showed that during the first hr numerous smooth vesicles and multivesicular bodies (mvb's) appear in the apical cytoplasm, while the number of coated vesicles and their relative total volume increase significantly 3 hr after stimulation.By infusion of ferritin in the pancreatic duct system in vivo and application of cytochemical techniques (osmium impregnation, electron microscope autoradiography and acid phosphatase cytochemistry) it could be established that after stimulated exocytotic secretion, redundant apical cell membrane is withdrawn by at least two routes: 1) During the initial rapid increase of the amount of apical cell membrane, withdrawal is accomplished by interiorization of luminal invaginations into smooth endocytotic vesicles, which in turn give rise to mvb's by infolding and subsequent fission of their limiting membrane. 2) Once the bulk of stored secretion granules has been discharged, endocytotic coated vesicles become gradually more prominent as carriers for redundant cell membrane. The contents of endocytotic structures ultimately become incorporated in residual bodies, suggesting lysosomal degradation of cell membrane prior to eventual reutilization.Coated vesicles also originate by pinching off from mature Golgi cisternae and condensing vacuoles. A possible function of the coated membranes in the concentration of exportable protein within forming secretory granules is discussed.     

Electron microscopy of stages of Isospora felis of the cat in the mesenteric lymph node of the mouse.

Stages of Isospora felis of the cat in the mesenteric lymph node of the mouse 25 days after oral inoculation with oocysts, have been described at the ultrastructural level. The organisms occurred singly within parasitophorous vacuoles in host cell cytoplasm and were sporozoite-like, having a large crystalloid body up to 5.5 mum in length posterior to the nucleus. The size and appearance of the parasitophorous vacuole varied. Some vacuoles contained numerous, small, electron dense granules about 30 nm in diameter. Because of the aggregation of granules and their arrangement within the parasitophorous vacuole, the impression was sometimes gained by light microscopy that parasites were surrounded by a sheath or cyst wall. However, a cyst wall was not present. In host cells, spherical, membrane-bound bodies with a homogeneous, electron dense core and a maximum diameter of 0.25 mum were filed along the limiting membrane of the parasitophorous vacuole. These extra-intestinal parasites were considered to be waiting stages, with a biological function similar to that of the tissue cyst stage of other general of isosporan coccidia.     

Ultrastructural cytochemical studies of plasma membrane phosphatase activities during the HeLa S3 cell cycle.

Alkaline phosphatase (AP), 5'-nucleotidase (5'N), Mg2+-activated ATPase (Mg-ATPase) and Ca2+-activated ATPase (Ca-ATPase) were studied in sychronized HeLa S3 cells with cytochemical methods and electron microscopy. It was found that AP activity, as determined by the deposition of lead phosphate reaction product (r.p.) was most active in mitotic (M), early and middle G1 cells, less active in late G1 and almost undetectable in S phase cells. Most AP enzyme activity was found to be associated with undulations (mainly microvilli) of the plasma membrane. Fluctuations and the redistribution of 5'N were also observed; the reaction for 5'N was positive in all phases of the cell cycle studied, it was strongest in M cells and in the majority of middle G1 cells. Mg-ATPase activity was present in the plasma membranes of cells throughout the cell cycle, but did not show noticeable fluctuations in activity and distribution. Ca-ATPase activity appeared in plasma membranes and in limited areas of cell nuclei but was evident only in S phase cells. The results of the present study confirm and extend previous biochemical observations and indicate that changes in membrane phosphate activities are associated with enzyme activity redistributions within the plasma membrane during the HeLa S3 cell cycle.     

ELECTRON MICROSCOPE OBSERVATIONS ON THE SURFACE ADENOSINE TRIPHOSPHATASE-LIKE ENZYMES OF HELA CELLS INFECTED WITH HERPES VIRUS

HeLa cells infected with herpes simplex virus have been examined in thin sections by electron microscopy after cytochemical staining for the presence of surface enzymes splitting adenosine triphosphate. As with uninfected HeLa cultures (18), the opaque enzyme reaction product was localized at the plasma membranes of about half the cells, tending to be present where there were microvilli and absent on smooth surfaces. Where mature extracellular herpes particles were found in association with cell membranes showing the enzyme activity, they were invariably likewise stained, and conversely, those mature particles which lay close against cells without reaction product at the surface were themselves free of it. Particles found budding into cytoplasmic vacuoles were also always without opaque deposit since this was never seen at vacuolar membranes, even in cells having the activity at the surface. The enzyme reaction product thus provided a marker indicating the manner in which the particles escape from cells and mature by budding out through cellular membranes, carrying, in the process, a portion of the latter on to themselves to form the outer viral limiting membrane. In some instances, virus particles were observed with more opaque material covering them than was present at the cell membrane with which they were associated. This finding has been taken as evidence for a physiological waxing and waning of surface enzyme activity of adenosine triphosphatase type. The fine structure of the mature extracellular virus as prepared here, using glutaraldehyde fixation, is also recorded. The observations and interpretations are discussed in full.     

Electron Microscopy of Tissue Culture Cells Infected with Brucella abortus

Thin sections of hamster kidney tissue cultures were examined by electron microscopy over a 7-day period after infection with Brucella abortus 3183. Numerous bacteria and structures resembling L-forms were present both intracellularly and extracellularly after the first 24 hr of infection. Most intracellular microorganisms were enclosed by a cytoplasmic membrane, but in a few instances no limiting membrane was detected. After 4 to 7 days, fewer microorganisms were present, and most normal-appearing bacteria were intracellular, particularly in antibiotic-treated cultures. Structures typical of Brucella L-forms were extracellular at the latter time intervals. Several structures were observed in cells from infected cultures whose relationship to the infecting organisms is not known. These consisted of various membranous structures within cytoplasmic vacuoles, myelin-like structures surrounding occasional intracellular organisms, and small bodies present within vacuoles and extracellularly. The latter structures observed throughout the experimental period appeared to occur more frequently as the duration of the infection increased.     

Immuno-Electron Microscopy of the Morphogenesis of Mumps Virus

The fine structure of mumps virus-infected chick embryo fibroblastic cells was examined sequentially after viral inoculation. Intracytoplasmic nucleoprotein strands, similar to those described for parainfluenza viruses, were detectable in small aggregates between 36 and 48 hr. The peripheral strands of this viral component lie beneath and along an antigenically altered bulging portion of the cell membrane. The outermost strands are consistently parallel to the differentiated segment of the plasma membrane, which is invariably associated with surface projections. As has been found with other myxoviruses, mumps virus replicates by budding from the cell surface. The virus particle, roughly spherical in shape, has a size ranging from 1,000 to 8,000 A. Filamentous forms are rarely observed in the present culture system. Ferritin-conjugated antibody specifically labels the cytoplasmic nucleoprotein, the modified cell membrane, and the virus particle. Intranuclear inclusions of low electron density and morphologically different from those described in measles virus-infected HeLa and amnion cells were observed in the nucleus of several infected cells. Immuno-electron microscopic observations suggest that the nucleoprotein synthesis rate exceeds that of cell membrane differentiation into viral envelope. This difference results in the accumulation of viral nucleoprotein in large intracytoplasmic masses which can be demonstrated by electron microscopy.